Human secreted proteins

ABSTRACT

The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diabetes mellitus and/or conditions related to diabetes. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

FIELD OF INVENTION

The present invention relates to human secreted proteins/polypeptides,and isolated nucleic acid molecules encoding said proteins/polypeptides,useful for detecting, preventing, diagnosing, prognosticating, treating,and/or ameliorating diabetes mellitus and conditions related thereto.Antibodies that bind these polypeptides are also encompassed by thepresent invention. Also encompassed by the invention are vectors, hostcells, and recombinant and synthetic methods for producing saidpolynucleotides, polypeptides, and/or antibodies. The invention furtherencompasses screening methods for identifying agonists and antagonistsof polynucleotides and polypeptides of the invention. The presentinvention further encompasses methods and compositions for inhibiting orenhancing the production and function of the polypeptides of the presentinvention.

BACKGROUND OF THE INVENTION

Over the past few decades, an increasing percentage of the populationhas become diabetic. Diabetes mellitus is categorized into two types:Type I, known as Insulin-Dependent Diabetes Mellitus (IDDM), or Type II,known as Non-Insulin-Dependent Diabetes Mellitus (NIDDM). IDDM is anautoinmmune disorder in which the insulin-secreting pancreatic betacells of the islets of Langerhans are destroyed. In these individuals,recombinant insulin therapy is employed to maintain glucose homeostasisand normal energy metabolism. NIDDM, on the other hand, is a polygenicdisorder with no one gene responsible for the progression of thedisease.

In NIDDM, insulin resistance eventually leads to the abolishment ofinsulin secretion resulting in insulin deficiency. Insulin resistance,at least in part, ensues from a block at the level of glucose uptake andphosphorylation in humans. Diabetics demonstrate a decrease inexpression in adipose tissue of insulin-receptor substrate 1 (“IRS1”)(Carvalho et al., FASEB J 13(15):2173-8 (1999)), glucose transporter 4(“GLUT4”) (Garvey et al., Diabetes 41(4):465-75 (1992)), and the novelabundant protein M gene transcript 1 (“apM1”) (Statnick et al., Int JExp Diabetes 1(2): 81-8 (2000)), as well as other as of yet unidentifiedfactors. Insulin deficiency in NIDDM leads to failure of normalpancreatic beta-cell function and eventually to pancreatic-beta celldeath.

Insulin affects fat, muscle, and liver. Insulin is the major regulatorof energy metabolism. Malfunctioning of any step(s) in insulin secretionand/or action can lead to many disorders, including for example thedysregulation of oxygen utilization, adipogenesis, glycogenesis,lipogenesis, glucose uptake, protein synthesis, thermogenesis, andmaintenance of the basal metabolic rate. This malfunctioning results indiseases and/or disorders that include, but are not limited to,hyperinsulinemia, insulin resistance, insulin deficiency, hyperglycemia,hyperlipidemia, hyperketonemia, and diabetes.

Numerous debilitating diabetes-related secondary effects include, butare not limited to, obesity, forms of blindness (cataracts and diabeticretinopathy), limb amputations, kidney failure, fatty liver, coronaryartery disease, and neuropathy.

Some of the current drugs used to treat insulin resistance and/ordiabetes (e.g., insulin secratogogues—sulfonylurea, insulinsensitizers—thiazolidenediones and metformin, and alpha-glucosidase andlipase inhibitors) are inadequate due to the dosage amounts andfrequency with which they have to be administered as a result of poorpharmacokinetic properties, the lack of effective control over bloodsugar levels, and potential side effects, among other reasons. DiabetesTherapeutic proteins in their native state or when recombinantlyproduced exhibit a rapid in vivo clearance. Typically, significantamounts of therapeutics are required to be effective during therapy. Inaddition, small molecules smaller than the 20 kDa range can be readilyfiltered through the renal tubules (glomerulus) leading todose-dependent nephrotoxicity. Therefore, there is a need forimprovement in treatment (e.g., a need for prolonging the effects oftherapeutics of diabetes and/or diabetes related conditions).

SUMMARY OF THE INVENTION

The present invention encompasses human secreted proteinsipolypeptides,and isolated nucleic acid molecules encoding said proteinsipolypeptides,useful for detecting, preventing, diagnosing, prognosticating, treating,and/or ameliorating diabetes mellitus and conditions related thereto.Antibodies that bind these polypeptides are also encompassed by thepresent invention; as are vectors, host cells, and recombinant andsynthetic methods for producing said polynucleotides, polypeptides,and/or antibodies. The invention further encompasses screening methodsfor identifying agonists and antagonists of polynucleotides andpolypeptides of the invention. The present invention also encompassesmethods and compositions for inhibiting or enhancing the production andfunction of the polypeptides of the present invention.

DETAILED DESCRIPTION

Polynucleotides and Polypeptides of the Invention

Description of Table 1A

Table 1A summarizes information concerning certain polypnucleotides andpolypeptides of the invention. The first column provides the gene numberin the application for each clone identifier. The second column providesa unique clone identifier, “Clone ID:”, for a cDNA clone related to eachcontig sequence disclosed in Table 1A. Third column, the cDNA Clonesidentified in the second column were deposited as indicated in the thirdcolumn (i.e. by ATCC Deposit No:Z and deposit date). Some of thedeposits contain multiple different clones corresponding to the samegene. In the fourth column, “Vector” refers to the type of vectorcontained in the corresponding cDNA Clone identified in the secondcolumn. In the fifth column, the nucleotide sequence identified as “NTSEQ ID NO:X” was assembled from partially homologous (“overlapping”)sequences obtained from the corresponding cDNA clone identified in thesecond column and, in some cases, from additional related cDNA clones.The overlapping sequences were assembled into a single contiguoussequence of high redundancy (usually three to five overlapping sequencesat each nucleotide position), resulting in a final sequence identifiedas SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the totalnumber of nucleotides in the contig sequence identified as SEQ ID NO:X.”The deposited clone may contain all or most of these sequences,reflected by the nucleotide position indicated as “5′ NT of Clone Seq.”(seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ IDNO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of theputative start codon (methionine) is identified as “5′ NT of StartCodon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:Xof the predicted signal sequence is identified as “5′ NT of First AA ofSignal Pep.” In the eleventh column, the translated amino acid sequence,beginning with the methionine, is identified as “AA SEQ ID NO:Y,”although other reading frames can also be routinely translated usingknown molecular biology techniques. The polypeptides produced by thesealternative open reading frames are specifically contemplated by thepresent invention.

In the twelfth and thirteenth columns of Table 1A, the first and lastamino acid position of SEQ ID NO:Y of the predicted signal peptide isidentified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In thefourteenth column, the predicted first amino acid position of SEQ IDNO:Y of the secreted portion is identified as “Predicted First AA ofSecreted Portion”. The amino acid position of SEQ ID NO:Y of the lastamino acid encoded by the open reading frame is identified in thefifteenth column as “Last AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X is useful for designing nucleic acid hybridization probesthat will detect nucleic acid sequences contained in SEQ ID NO:X or thecDNA contained in the deposited clone. These probes will also hybridizeto nucleic acid molecules in biological samples, thereby enabling avariety of forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used, for example, togenerate antibodies which bind specifically to proteins containing thepolypeptides and the secreted proteins encoded by the cDNA clonesidentified in Table 1A and/or elsewhere herein

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X, and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1A. Thenucleotide sequence of each deposited plasmid can readily be determinedby sequencing the deposited plasmid in accordance with known methods

The predicted amino acid sequence can then be verified from suchdeposits. Moreover, the amino acid sequence of the protein encoded by aparticular plasmid can also be directly determined by peptide sequencingor by expressing the protein in a suitable host cell containing thedeposited human cDNA, collecting the protein, and determining itssequence.

Also provided in Table 1A is the name of the vector which contains thecDNA plasmid. Each vector is routinely used in the art. The followingadditional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Phagemid pBS may be excised fromthe Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excisedfrom the Zap Eipress vector. Both phagemids may be transformed into E.coli strain XL-1 Blue, also available from Stratagene

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, wereobtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md.20897. All Sport vectors contain an ampicillin resistance gene and maybe transformed into E. coli strain DH1OB, also available from LifeTechnologies. See, for instance, Gruber, C. E., et al., Focus 15:59(1993). Vector lafmid BA (Bento Soares, Columbia University, New York,N.Y.) contains an ampicillin resistance gene and can be transformed intoE. coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH1OB, available from Life Technologies. See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,BiolTechnology 9: (1991).

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). Thecorresponding gene can be isolated in accordance with known methodsusing the sequence information disclosed herein. Such methods include,but are not limited to, preparing probes or primers from the disclosedsequence and identifying or amplifying the corresponding gene fromappropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X and SEQ ID NO:Y using information from thesequences disclosed herein or the clones deposited with the ATCC. Forexample, allelic variants and/or species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:Xand/or a cDNA contained in ATCC Deposit No.Z. The present invention alsoprovides a polypeptide comprising, or alternatively, consisting of, thepolypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ IDNO:X, and/or a polypeptide encoded by a cDNA contained in ATCC depositNo.Z. Polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y, apolypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by thecDNA contained in ATCC Deposit No.Z, are also encompassed by theinvention. The present invention further encompasses a polynucleotidecomprising, or alternatively consisting of the complement of the nucleicacid sequence of SEQ ID NO:X, and/or the complement of the coding strandof the cDNA contained in ATCC Deposit No.Z.

Description of Table 1B (Comprised of Tables 1B.1 and 1B.2)

Table 1B.1 and Table 1B.2 summarize some of the polynucleotidesencompassed by the invention (including cDNA clones related to thesequences (Clone ID:), contig sequences (contig identifier (Contig ID:)and contig nucleotide sequence identifiers (SEQ ID NO:X)) and furthersummarizes certain characteristics of these polynucleotides and thepolypeptides encoded thereby. The first column of Tables 1B. 1 and 1B.2provide the gene numbers in the application for each clone identifier.The second column of Tables 1B.1 and 1B.2 provide unique cloneidentifiers, “Clone ID:”, for cDNA clones related to each contigsequence disclosed in Table 1A and/or Table 1B. The third column ofTables 1B.1 and 1B.2 provide unique contig identifiers, “Contig ID:” foreach of the contig sequences disclosed in these tables. The fourthcolumn of Tables 1B.1 and 1B.2 provide the sequence identifiers, “SEQ IDNO:X”, for each of the contig sequences disclosed in Table 1A and/or 1B.

Table 1B.1

The fifth column of Table 1B.1, “ORF (From-To)”, provides the location(i.e., nucleotide position numbers) within the polynucleotide sequenceof SEQ ID NO:X that delineates the preferred open reading frame (ORF)that encodes the amino acid sequence shown in the sequence listing andreferenced in Table 1B.1 as SEQ ID NO:Y (column 6). Column 7 of Table1B.1 lists residues comprising predicted epitopes contained in thepolypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y).Identification of potential immunogenic regions was performed accordingto the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988));specifically, the Genetics Computer Group (GCG) implementation of thisalgorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Packagev10.0, Genetics Computer Group (GCG), Madison, Wis.). This methodreturns a measure of the probability that a given residue is found onthe surface of the protein. Regions where the antigenic index score isgreater than 0.9 over at least 6 amino acids are indicated in Table 1B.1as “Predicted Epitopes”. In particular embodiments, polypeptides of theinvention comprise, or alternatively consist of, one, two, three, four,five or more of the predicted epitopes described in Table 1B.1. It willbe appreciated that depending on the analytical criteria used to predictantigenic determinants, the exact address of the determinant may varyslightly. Column 8 of Table 1B.1 (“Cytologic Band”) provides thechromosomal location of polynucleotides corresponding to SEQ ID NO:X.Chromosomal location was determined by finding exact matches to EST andcDNA sequences contained in the NCBI (National Center for BiotechnologyInformation) UniGene database. Given a presumptive chromosomal location,disease locus association was determined by comparison with the MorbidMap, derived from Online Mendelian Inheritance in Man (Online MendelianInheritance in Man, OMIM™. McKusick-Nathans Institute for GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.) 2000. World Wide Web URL: http:/www.ncbi.nlm.nih.gov/omim/). If theputative chromosomal location of the Query overlaps with the chromosomallocation of a Morbid Map entry, an OMIM identification number isdisclosed in Table 1B.1, column 9 labeled “OMIM Disease Reference(s)”. Akey to the OMIM reference identification numbers is provided in Table 5.

Table 1B.2

Column 5 of Table 1B.2, “Tissue Distribution” shows the expressionprofile of tissue, cells, and/or cell line libraries which express thepolynucleotides of the invention. The first code number shown in Table1B.2 column 5 (preceding the colon), represents the tissue/cell sourceidentifier code corresponding to the key provided in Table 4. Expressionof these polynucleotides was not observed in the other tissues and/orcell libraries tested. The second number in column 5 (following thecolon), represents the number of times a sequence corresponding to thereference polynucleotide sequence (e.g., SEQ ID NO:X) was identified inthe corresponding tissue/cell source. Those tissue/cell sourceidentifier codes in which the first two letters are “AR” designateinformation generated using DNA array technology. Utilizing thistechnology, cDNAs were amplified by PCR and then transferred, induplicate, onto the array. Gene expression was assayed throughhybridization of first strand cDNA probes to the DNA array. cDNA probeswere generated from total RNA extracted from a variety of differenttissues and cell lines. Probe synthesis was performed in the presence of³³P dCTP, using oligo(dT) to prime reverse transcription. Afterhybridization, high stringency washing conditions were employed toremove non-specific hybrids from the array. The remaining signal,emanating from each gene target, was measured using a Phosphorimager.Gene expression was reported as Phosphor Stimulating Luminescence (PSL)which reflects the level of phosphor signal generated from the probehybridized to each of the gene targets represented on the array. A localbackground signal subtraction was performed before the total signalgenerated from each array was used to normalize gene expression betweenthe different hybridizations. The value presented after “[array code]:”represents the mean of the duplicate values, following backgroundsubtraction and probe normalization. One of skill in the art couldroutinely use this information to identify normal and/or diseasedtissue(s) which show a predominant expression pattern of thecorresponding polynucleotide of the invention or to identifypolynucleotides which show predominant and/or specific tissue and/orcell expression.

Description of Table 1C

Table 1C summarizes additional polynucleotides encompassed by theinvention (including cDNA clones related to the sequences (Clone ID:),contig sequences (contig identifier (Contig ID:) contig nucleotidesequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ IDNO:B). The first column provides a unique clone identifier, “Clone ID:”,for a cDNA clone related to each contig sequence. The second columnprovides the sequence identifier, “SEQ ID NO:X”, for each contigsequence. The third column provides a unique contig identifier, “ContigID:” for each contig sequence. The fourth column, provides a BACidentifier “BAC ID NO:A” for the BAC clone referenced in thecorresponding row of the table. The fifth column provides the nucleotidesequence identifier, “SEQ ID NO:B” for a fragment of the BAC cloneidentified in column four of the corresponding row of the table. Thesixth column, “Exon From-To”, provides the location (i.e., nucleotideposition numbers) within the polynucleotide sequence of SEQ ID NO:Bwhich delineate certain polynucleotides of the invention that are alsoexemplary members of polynucleotide sequences that encode polypeptidesof the invention (e.g., polypeptides containing amino acid sequencesencoded by the polynucleotide sequences delineated in column six, andfragments and variants thereof).

Description of Table 1D

Table 1D: In preferred embodiments, the present invention encompasses amethod of detecting, preventing, diagnosing, prognosticating, treating,and/or ameliorating diabetes mellitus; comprising administering to apatient in which such treatment, prevention, or amelioration is desireda protein, nucleic acid, or antibody of the invention (or fragment orvariant thereof) represented by Table 1A, Table 1B, and Table 1C, in anamount effective to detect, prevent, diagnose, prognosticate, treat,and/or ameliorate the disease or disorder.

As indicated in Table 1D, the polynucleotides, polypeptides, agonists,or antagonists of the present invention (including antibodies) can beused in assays to test for one or more biological activities. If thesepolynucleotides and polypeptides do exhibit activity in a particularassay, it is likely that these molecules may be involved in the diseasesassociated with the biological activity. Thus, the polynucleotides orpolypeptides, or agonists or antagonists thereof (including antibodies)could be used to treat the associated disease.

Table 1D provides information related to biological activities forpolynucleotides and polypeptides of the invention (including antibodies,agonists, and/or antagonists thereof). Table 1D also providesinformation related to assays which may be used to test polynucleotidesand polypeptides of the invention (including antibodies, agonists,and/or antagonists thereof) for the corresponding biological activities.The first column (“Gene No.”) provides the gene number in theapplication for each clone identifier. The second column (“cDNA CloneID:”) provides the unique clone identifier for each clone as previouslydescribed and indicated in Tables 1A, 1B, and 1C. The third column (“AASEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number forpolypeptide sequences encoded by the corresponding cDNA clones (also asindicated in Tables 1A, 1B, and 2). The fourth column (“BiologicalActivity”) indicates a biological activity corresponding to theindicated polypeptides (or polynucleotides encoding said polypeptides).The fifth column (“Exemplary Activity Assay”) further describes thecorresponding biological activity and provides information pertaining tothe various types of assays which may be performed to test, demonstrate,or quantify the corresponding biological activity. Table 1D describesthe use of FMAT technology, inter alia, for testing or demonstratingvarious biological activities. Fluorometric microvolume assay technology(FMAT) is a fluorescence-based system which provides a means to performnonradioactive cell- and bead-based assays to detect activation of cellsignal transduction pathways. This technology was designed specificallyfor ligand binding and immunological assays. Using this technology,fluorescent cells or beads at the bottom of the well are detected aslocalized areas of concentrated fluorescence using a data processingsystem. Unbound flurophore comprising the background signal is ignored,allowing for a wide variety of homogeneous assays. FMAT technology maybe used for peptide ligand binding assays, immunofluorescence,apoptosis, cytotoxicity, and bead-based immunocapture assays. See,Miraglia S et. al., “Homogeneous cell and bead based assays forhighthroughput screening using flourometric microvolume assaytechnology,” Journal of Biomolecular Screening; 4:193-204 (1999). Inparticular, FMAT technology may be used to test, confirm, and/oridentify the ability of polypeptides (including polypeptide fragmentsand variants) to activate signal transduction pathways. For example,FMAT technology may be used to test, confirm, and/or identify theability of polypeptides to upregulate production of immunomodulatoryproteins (such as, for example, interleukins, GM-CSF, Rantes, and TumorNecrosis factors, as well as other cellular regulators (e.g. insulin)).

Table 1D also describes the use of kinase assays for testing,demonstrating, or quantifying biological activity. In this regard, thephosphorylation and de-phosphorylation of specific amino acid residues(e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteinsprovides a fast, reversible means for activation and de-activation ofcellular signal transduction pathways. Moreover, cell signaltransduction via phosphorylation/de-phosphorylation is crucial to theregulation of a wide variety of cellular processes (e.g. proliferation,differentiation, migration, apoptosis, etc.). Accordingly, kinase assaysprovide a powerful tool useful for testing, confirming, and/oridentifying polypeptides (including polypeptide fragments and variants)that mediate cell signal transduction events via proteinphosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R.“Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).

Description of Table 2

Table 2 summarizes homology and features of some of the polypeptides ofthe invention. The first column provides a unique clone identifier,“Clone ID:”, corresponding to a cDNA clone disclosed in Table 1A orTable 1B. The second column provides the unique contig identifier,“Contig ID:” corresponding to contigs in Table 1B and allowing forcorrelation with the information in Table 1B. The third column providesthe sequence identifier, “SEQ ID NO:X”, for the contig polynucleotidesequence. The fourth column provides the analysis method by which thehomology/identity disclosed in the Table was determined. Comparisonswere made between polypeptides encoded by the polynucleotides of theinvention and either a non-redundant protein database (herein referredto as “NR”), or a database of protein families (herein referred to as“PFAM”) as further described below. The fifth column provides adescription of the PFAM/NR hit having a significant match to apolypeptide of the invention. Column six provides the accession numberof the PFAM/NR hit disclosed in the fifth column. Column seven,“Score/Percent Identity”, provides a quality score or the percentidentity, of the hit disclosed in columns five and six. Columns 8 and 9,“NT From” and “NT To” respectively, delineate the polynucleotides in“SEQ ID NO:X” that encode a polypeptide having a significant match tothe PFAM/NR database as disclosed in the fifth and sixth columns. Inspecific embodiments polypeptides of the invention comprise, oralternatively consist of, an amino acid sequence encoded by apolynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, orfragments or variants thereof.

Description of Table 3

Table 3 provides polynucleotide sequences that may be disclaimedaccording to certain embodiments of the invention. The first columnprovides a unique clone identifier, “Clone ID”, for a cDNA clone relatedto contig sequences disclosed in Table 1B. The second column providesthe sequence identifier, “SEQ ID NO:X”, for contig sequences disclosedin Table 1A and/or Table 1B. The third column provides the unique contigidentifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourthcolumn provides a unique integer ‘a’ where ‘a’ is any integer between 1and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth columnprovides a unique integer ‘b’ where ‘b’ is any integer between 15 andthe final nucleotide of SEQ ID NO:X, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:X, and where bis greater than or equal to a +14. For each of the polynucleotides shownas SEQ ID NO:X, the uniquely defined integers can be substituted intothe general formula of a-b, and used to describe polynucleotides whichmay be preferably excluded from the invention. In certain embodiments,preferably excluded from the invention are at least one, two, three,four, five, ten, or more of the polynucleotide sequence(s) having theaccession number(s) disclosed in the sixth column of this Table(including for example, published sequence in connection with aparticular BAC clone). In further embodiments, preferably excluded fromthe invention are the specific polynucleotide sequence(s) contained inthe clones corresponding to at least one, two, three, four, five, ten,or more of the available material having the accession numbersidentified in the sixth column of this Table (including for example, theactual sequence contained in an identified BAC clone).

Description of Table 4

Table 4 provides a key to the tissue/cell source identifier codedisclosed in Table 1B.2, column 5. Column 1 provides the tissue/cellsource identifier code disclosed in Table 1B.2, Column 5. Columns 2-5provide a description of the tissue or cell source. Note that“Description” and “Tissue” sources (i.e. columns 2 and 3) having theprefix “a_” indicates organs, tissues, or cells derived from “adult”sources. Codes corresponding to diseased tissues are indicated in column6 with the word “disease.” The use of the word “disease” in column 6 isnon-limiting. The tissue or cell source may be specific (e.g. aneoplasm), or may be disease-associated (e.g., a tissue sample from anormal portion of a diseased organ). Furthermore, tissues and/or cellslacking the “disease” designation may still be derived from sourcesdirectly or indirectly involved in a disease state or disorder, andtherefore may have a further utility in that disease state or disorder.In numerous cases where the tissue/cell source is a library, column 7identifies the vector used to generate the library.

Description of Table 5

Table 5 provides a key to the OMIM reference identification numbersdisclosed in Table 1B.1, column 9. OMIM reference identification numbers(Column 1) were derived from Online Mendelian Inheritance in Man (OnlineMendelian Inheritance in Man, OMIM. McKusick-Nathans Institute forGenetic Medicine, Johns Hopkins University (Baltimore, Md.) and NationalCenter for Biotechnology Information, National Library of Medicine,(Bethesda, Md.) 2000. World Wide Web URL:http://www.ncbi.nlm.nih.govlomim/). Column 2 provides diseasesassociated with the cytologic band disclosed in Table 1B.1, column 8, asdetermined using the Morbid Map database.

Description of Table 6

Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCCdesignation numbers of deposits made with the ATCC in connection withthe present application. These deposits were made in addition to thosedescribed in the Table 1A.

Description of Table 7

Table 7 shows the cDNA libraries sequenced, and ATCC designation numbersand vector information relating to these cDNA libraries.

The first column shows the first four letters indicating the Libraryfrom which each library clone was derived. The second column indicatesthe catalogued tissue description for the corresponding libraries. Thethird column indicates the vector containing the corresponding clones.The fourth column shows the ATCC deposit designation for each librayclone as indicated by the deposit information in Table 6.

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or MnRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genornic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, a “secreted” protein refers to those proteinscapable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as thoseproteins released into the extracellular space without necessarilycontaining a signal sequence. If the secreted protein is released intothe extracellular space, the secreted protein can undergo extracellularprocessing to produce a “mature” protein. Release into the extracellularspace can occur by many mechanisms, including exocytosis and proteolyticcleavage.

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence encoding SEQ ID NO:Y or a fragment or variant thereof(e.g., the polypeptide delinated in columns fourteen and fifteen ofTable 1A); a nucleic acid sequence contained in SEQ ID NO:X (asdescribed in column 5 of Table 1A and/or column 3 of Table 1B) or thecomplement thereof; a cDNA sequence contained in Clone ID: (as describedin column 2 of Table 1A and/or Table 1B and contained within a librarydeposited with the ATCC); a nucleotide sequence encoding the polypeptideencoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6(EXON From-To) of Table 1C or a fragment or variant thereof; or anucleotide coding sequence in SEQ ID NO:B as defined in column 6 ofTable 1C or the complement thereof. For example, the polynucleotide cancontain the nucleotide sequence of the full length cDNA sequence,including the 5′ and 3′ untranslated sequences, the coding region, aswell as fragments, epitopes, domains, and variants of the nucleic acidsequence. Moreover, as used herein, a “polypeptide” refers to a moleculehaving an amino acid sequence encoded by a polynucleotide of theinvention as broadly defined (obviously excluding poly-Phenylalanine orpoly-Lysine peptide sequences which result from translation of a polyAtail of a sequence corresponding to a cDNA).

In the present invention, “SEQ ID NO:X” was often generated byoverlapping sequences contained in multiple clones (contig analysis). Arepresentative clone containing all or most of the sequence for SEQ IDNO:X is deposited at Human Genome Sciences, Inc. (HGS) in a cataloguedand archived library. As shown, for example, in column 2 of Table 1B,each clone is identified by a cDNA Clone ID (identifier generallyreferred to herein as Clone ID:). Each Clone ID is unique to anindividual clone and the Clone ID is all the information needed toretrieve a given clone from the HGS library. Table 7 provides a list ofthe deposited cDNA libraries. One can use the Clone ID: to determine thelibrary source by reference to Tables 6 and 7. Table 7 lists thedeposited cDNA libraries by name and links each library to an ATCCDeposit. Library names contain four characters, for example, “HTWE.” Thename of a cDNA clone (Clone ID) isolated from that library begins withthe same four characters, for example “HTWEP07”. As mentioned below,Table 1A and/or Table 1B correlates the Clone ID names with SEQ ID NO:X.Thus, starting with an SEQ ID NO:X, one can use Tables 1A, 1B, 6, 7, and9 to determine the corresponding Clone ID, which library it came fromand which ATCC deposit the library is contained in. Furthermore, it ispossible to retrieve a given cDNA clone from the source library bytechniques known in the art and described elsewhere herein. The ATCC islocated at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.The ATCC deposits were made pursuant to the terms of the Budapest Treatyon the international recognition of the deposit of microorganisms forthe purposes of patent procedure.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, or the complementthereof (e.g., the complement of any one, two, three, four, or more ofthe polynucleotide fragments described herein), the polynucleotidesequence delineated in columns 7 and 8 of Table 1A or the complementthereof, the polynucleotide sequence delineated in columns 8 and 9 ofTable 2 or the complement thereof, and/or cDNA sequences contained inClone ID: (e.g., the complement of any one, two, three, four, or more ofthe polynucleotide fragments, or the cDNA clone within the pool of cDNAclones deposited with the ATCC, described herein), and/or thepolynucleotide sequence delineated in column 6 of Table 1C or thecomplement thereof. “Stringent hybridization conditions” refers to anovernight incubation at 42 degree C. in a solution comprising 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCI; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

“SEQ ID NO:X” refers to a polynucleotide sequence described in column 5of Table 1A, while “SEQ ID NO:Y” refers to a polypeptide sequencedescribed in column 10 of Table 1A. SEQ ID NO:X is identified by aninteger specified in column 6 of Table 1A. The polypeptide sequence SEQID NO:Y is a translated open reading frame (ORF) encoded bypolynucleotide SEQ ID NO:X. The polynucleotide sequences are shown inthe sequence listing immediately followed by all of the polypeptidesequences. Thus, a polypeptide sequence corresponding to polynucleotidesequence SEQ ID NO:2 is the first polypeptide sequence shown in thesequence listing. The second polypeptide sequence corresponds to thepolynucleotide sequence shown as SEQ ID NO:3, and so on.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid sidehains and the amino or carboxyl termini. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formnation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

“SEQ ID NO:X” refers to a polynucleotide sequence described, forexample, in Tables 1A, Table 1B, or Table 2, while “SEQ ID NO:Y” refersto a polypeptide sequence described in column 11 of Table 1A and or ofTable 1B. SEQ ID NO:X is identified by an integer specified in column 4of Table 1B. The polypeptide sequence SEQ ID NO:Y is a translated openreading frame (ORF) encoded by polynucleotide SEQ ID NO:X. “Clone ID:”refers to a cDNA clone described in column 2 of Table 1A and/or 1B.

“A polypeptide having functional activity” refers to a polypeptidecapable of displaying one or more known functional activities associatedwith a full-length (complete) protein. Such functional activitiesinclude, but are not limited to, biological activity (e.g. activityuseful in treating, preventing and/or ameliorating diabetes mellitus),antigenicity (ability to bind [or compete with a polypeptide forbinding] to an anti-polypeptide antibody), immunogenicity (ability togenerate antibody which binds to a specific polypeptide of theinvention), ability to form multimers with polypeptides of theinvention, and ability to bind to a receptor or ligand for apolypeptide.

The polypeptides of the invention can be assayed for functional activity(e.g. biological activity) using or routinely modifying assays known inthe art, as well as assays described herein. Specifically, one of skillin the art may routinely assay secreted polypeptides (includingfragments and variants) of the invention for activity using assays asdescribed in the examples section below.

“A polypeptide having biological activity” refers to a polypeptideexhibiting activity similar to, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention).

Tables

Table 1A

Table 1A summarizes information concerning certain polypnucleotides andpolypeptides of the invention. The first column provides the gene numberin the application for each clone identifier. The second column providesa unique clone identifier, “Clone ID:”, for a cDNA clone related to eachcontig sequence disclosed in Table 1A. Third column, the cDNA Clonesidentified in the second column were deposited as indicated in the thirdcolumn (i.e. by ATCC Deposit No:Z and deposit date). Some of thedeposits contain multiple different clones corresponding to the samegene. In the fourth column, “Vector” refers to the type of vectorcontained in the corresponding cDNA Clone identified in the secondcolumn. In the fifth column, the nucleotide sequence identified as “NTSEQ ID NO:X” was assembled from partially homologous (“overlapping”)sequences obtained from the corresponding cDNA clone identified in thesecond column and, in some cases, from additional related cDNA clones.The overlapping sequences were assembled into a single contiguoussequence of high redundancy (usually three to five overlapping sequencesat each nucleotide position), resulting in a final sequence identifiedas SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the totalnumber of nucleotides in the contig sequence identified as SEQ ID NO:X.”The deposited clone may contain all or most of these sequences,reflected by the nucleotide position indicated as “5′ NT of Clone Seq.”(seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ IDNO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of theputative start codon (methionine) is identified as “5′ NT of StartCodon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:Xof the predicted signal sequence is identified as “5′ NT of First AA ofSignal Pep.” In the eleventh column, the translated amino acid sequence,beginning with the methionine, is identified as “AA SEQ ID NO:Y,”although other reading frames can also be routinely translated usingknown molecular biology techniques. The polypeptides produced by thesealternative open reading frames are specifically contemplated by thepresent invention.

In the twelfth and thirteenth columns of Table 1A, the first and lastamino acid position of SEQ ID NO:Y of the predicted signal peptide isidentified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In thefourteenth column, the predicted first amino acid position of SEQ IDNO:Y of the secreted portion is identified as “Predicted First AA ofSecreted Portion”. The amino acid position of SEQ ID NO:Y of the lastamino acid encoded by the open reading frame is identified in thefifteenth column as “Last AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X is useful for designing nucleic acid hybridization probesthat will detect nucleic acid sequences contained in SEQ ID NO:X or thecDNA contained in the deposited clone. These probes will also hybridizeto nucleic acid molecules in biological samples, thereby enabling avariety of forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used, for example, togenerate antibodies which bind specifically to proteins containing thepolypeptides and the secreted proteins encoded by the cDNA clonesidentified in Table 1A and/or elsewhere herein

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X, and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1A. Thenucleotide sequence of each deposited plasmid can readily be determinedby sequencing the deposited plasmid in accordance with known methods

The predicted amino acid sequence can then be verified from suchdeposits. Moreover, the amino acid sequence of the protein encoded by aparticular plasmid can also be directly determined by peptide sequencingor by expressing the protein in a suitable host cell containing thedeposited human cDNA, collecting the protein, and determining itssequence.

Also provided in Table 1A is the name of the vector which contains thecDNA plasmid. Each vector is routinely used in the art. The followingadditional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Phagemid pBS may be excised fromthe Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excisedfrom the Zap Express vector. Both phagemids may be transformed into E.coli strain XL-1 Blue, also available from Stratagene

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, wereobtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md.20897. All Sport vectors contain an ampicillin resistance gene and maybe transformed into E. coli strain DHIOB, also available from LifeTechnologies. See, for instance, Gruber, C. E., et al., Focus 15:59(1993). Vector lafmid BA (Bento Soares, Columbia University, New York,N.Y.) contains an ampicillin resistance gene and can be transformed intoE. coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). Thecorresponding gene can be isolated in accordance with known methodsusing the sequence information disclosed herein. Such methods include,but are not limited to, preparing probes or primers from the disclosedsequence and identifying or amplifying the corresponding gene fromappropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X and SEQ ID NO:Y using information from thesequences disclosed herein or the clones deposited with the ATCC. Forexample, allelic variants and/or species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:Xand/or a cDNA contained in ATCC Deposit No.Z. The present invention alsoprovides a polypeptide comprising, or alternatively, consisting of, thepolypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ IDNO:X, and/or a polypeptide encoded by a cDNA contained in ATCC depositNo.Z. Polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y, apolypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by thecDNA contained in ATCC Deposit No.Z, are also encompassed by theinvention. The present invention further encompasses a polynucleotidecomprising, or alternatively consisting of the complement of the nucleicacid sequence of SEQ ID NO:X, and/or the complement of the coding strandof the cDNA contained in ATCC Deposit No.Z. TABLE 1A NT 5′ NT AA FirstLast ATCC SEQ 5′ NT 3′ NT of First SEQ AA AA First AA Last Deposit IDTotal of of 5′ NT AA of ID of of of AA Gene cDNA No: Z and NO: NT CloneClone of Start Signal NO: Sig Sig Secreted of No. Clone ID Date Vector XSeq. Seq. Seq. Codon Pep Y Pep Pep Portion ORF 1 H6EDM64 203959 Uni-ZAPXR 11 2610 1275 2377 1448 1448 205 1 6 Apr. 26, 1999 2 H6EEU40 203917Uni-ZAP XR 12 951 1 951 175 175 206 1 27 28 47 Apr. 8, 1999 3 HACAB68203917 Uni-ZAP XR 13 1300 1 1300 135 135 207 1 26 27 78 Apr. 8, 1999 4HACBJ56 203979 Uni-ZAP XR 14 888 1 888 250 208 1 9 10 25 Apr. 29, 1999 5HADMB15 203979 pBluescript 15 330 1 330 238 209 1 11 12 20 Apr. 29, 19996 HAGDW20 203917 Uni-ZAP XR 16 1284 1 1284 238 238 210 1 16 17 17 Apr.8, 1999 7 HAGEQ79 203917 Uni-ZAP XR 17 785 1 785 515 515 211 1 11 Apr.8, 1999 8 HAJBV67 PTA-181 pCMVSport 18 2536 1 2536 605 605 212 1 19 20359 Jun. 7, 1999 3.0 9 HAJCH70 203917 pCMVSport 19 2182 1 2182 284 284213 1 32 33 38 Apr. 8, 1999 3.0 10 HAOAG15 203979 pSport1 20 5143 7 48028 214 1 22 23 1167 Apr. 29, 1999 11 HATCD80 203917 Uni-ZAP XR 21 1809 951809 296 296 215 1 23 24 37 Apr. 8, 1999 12 HATCI03 203917 Uni-ZAP XR 22934 1 934 271 271 216 1 17 Apr. 8, 1999 13 HBAGD86 203917 pSport1 231713 293 1596 521 521 217 1 18 19 19 Apr. 8, 1999 14 HBHAA05 203917Uni-ZAP XR 24 690 1 690 110 218 1 16 17 58 Apr. 8, 1999 15 HBHAA81203959 Uni-ZAP XR 25 1647 1 1647 28 28 219 1 24 25 203 Apr. 26, 1999 16HBIAA59 203917 Uni-ZAP XR 26 2392 1612 2392 1877 1877 220 1 15 16 136Apr. 8, 1999 17 HBJAC40 203979 Uni-ZAP XR 27 1767 184 1729 329 221 1 13Apr. 29, 1999 18 HBJCR46 203917 Uni-ZAP XR 28 3208 2270 3202 589 589 2221 1 2 733 Apr. 8, 1999 19 HBJDW56 203917 Uni-ZAP XR 29 637 1 637 121 2231 8 Apr. 8, 1999 20 HBJEL16 203979 Uni-ZAP XR 30 750 1 750 115 115 224 124 25 36 Apr. 29, 1999 21 HBJIG20 PTA-181 Uni-ZAP XR 31 637 1 637 321225 1 16 17 77 Jun. 7, 1999 22 HBJKD16 203979 Uni-ZAP XR 32 1629 1 162978 78 226 1 18 19 31 Apr. 29, 1999 23 HBMTY48 203917 Uni-ZAP XR 33 18911 1891 660 660 227 1 36 37 94 Apr. 8, 1999 24 HBMUH74 PTA-181 Uni-ZAP XR34 726 1 726 344 344 228 1 13 14 28 Jun. 7, 1999 25 HBQAB79 203917Lambda ZAP 35 1331 1 1331 190 190 229 1 11 Apr. 8, 1999 II 26 HBXCX15203917 ZAP Express 36 1219 1 1219 1148 230 1 1 Apr. 8, 1999 27 HCDCY76203917 Uni-ZAP XR 37 1392 628 1392 860 231 1 17 18 35 Apr. 8, 1999 28HCEDR26 203917 Uni-ZAP XR 38 1419 1 1419 177 177 232 1 26 27 55 Apr. 8,1999 29 HCEEU18 203917 Uni-ZAP XR 39 1229 1 1229 209 209 233 1 30 31 43Apr. 8, 1999 30 HCEGX05 203917 Uni-ZAP XR 40 1305 1 1305 237 237 234 115 Apr. 8, 1999 31 HCFLN88 203917 pSport1 41 1434 1 1434 101 101 235 116 17 25 Apr. 8, 1999 32 HCHAB84 203979 pSport1 42 1359 62 1359 304 2361 23 24 147 Apr. 29, 1999 33 HCMSX51 203917 Uni-ZAP XR 43 2253 334 2190539 237 1 31 32 80 Apr. 8, 1999 34 HCNCO11 203917 Lambda ZAP 44 746 1746 101 101 238 1 14 Apr. 8, 1999 II 35 HCNSD29 PTA-181 pBluescript 451728 1031 1633 1145 1145 239 1 19 20 31 Jun. 7, 1999 36 HCQBH72 203917Lambda ZAP 46 1796 776 1796 31 31 240 1 25 26 47 Apr. 8, 1999 II 37HCQCC96 203979 Lambda ZAP 47 2166 632 1455 782 782 241 1 20 21 45 Apr.29, 1999 II 38 HCQCJ56 203917 Lambda ZAP 48 1287 1 1287 728 242 1 1 Apr.8, 1999 II 39 HCUDD64 203917 ZAP Express 49 402 150 389 256 256 243 1 3536 49 Apr. 8, 1999 40 HCWAE64 203917 ZAP Express 50 471 1 471 410 244 15 Apr. 8, 1999 41 HDPDJ58 203960 pCMVSport 51 1997 1 1997 279 279 245 120 Apr. 26, 1999 3.0 42 HDPFU43 203960 pCMVSport 52 1904 1 1889 220 220246 1 28 29 52 Apr. 26, 1999 3.0 43 HDPGE24 203960 pCMVSport 53 2625 12625 173 173 247 1 11 12 73 Apr. 26, 1999 3.0 44 HDPIU94 203960pCMVSport 54 2196 21 2196 208 208 248 1 21 22 23 Apr. 26, 1999 3.0 45HDPIY31 PTA-793 pCMVSport 55 1978 1 1978 268 268 249 1 16 17 35 Sep. 27,1999 3.0 46 HDPOC24 203960 pCMVSport 56 1777 302 1725 418 418 250 1 2324 133 Apr. 26, 1999 3.0 47 HDPPD93 203960 pCMVSport 57 701 1 701 28 28251 1 12 Apr. 26, 1999 3.0 48 HDPPQ30 203960 pCMVSport 58 1063 1 1063220 220 252 1 22 23 38 Apr. 26, 1999 3.0 49 HDQHM36 PTA-181 pCMVSport 591547 1 1547 129 129 253 1 18 19 48 Jun. 7, 1999 3.0 50 HDTFX18 203960pCMVSport 60 678 1 678 164 164 254 1 16 17 20 Apr. 26, 1999 2.0 51HE2CM39 203960 Uni-ZAP XR 61 566 1 566 10 255 1 13 Apr. 26, 1999 52HE2HC60 203960 Uni-ZAP XR 62 1569 236 1569 273 273 256 1 16 17 39 Apr.26, 1999 53 HE2PO93 203960 Uni-ZAP XR 63 1323 638 1323 770 770 257 1 2728 42 Apr. 26, 1999 54 HE6FU11 203979 Uni-ZAP XR 64 2000 1 1994 145 145258 1 26 27 226 Apr. 29, 1999 55 HE6FV29 203960 Uni-ZAP XR 65 1526 11526 210 210 259 1 18 19 33 Apr. 26, 1999 56 HE8TY46 PTA- Uni-ZAP XR 662204 1400 2204 1413 1413 260 1 18 19 187 1838 May 9, 2000 57 HE9EA10203960 Uni-ZAP XR 67 2114 1 2111 212 261 1 28 29 78 Apr. 26, 1999 58HEBCY54 203960 Uni-ZAP XR 68 1189 1 1189 172 172 262 1 24 25 118 Apr.26, 1999 59 HEBFR46 203979 Uni-ZAP XR 69 1304 1 1304 200 200 263 1 26 2729 Apr. 29, 1999 60 HEBGE07 203960 Uni-ZAP XR 70 1867 1 1867 106 106 2641 25 26 42 Apr. 26, 1999 61 HEGAU15 203960 Uni-ZAP XR 71 1125 1 1125 5959 265 1 30 31 34 Apr. 26, 1999 62 HETCI16 203979 Uni-ZAP XR 72 2285 732285 237 237 266 1 27 28 40 Apr. 29, 1999 63 HFCEI04 203960 Uni-ZAP XR73 887 1 887 136 267 1 17 18 42 Apr. 26, 1999 64 HFCFE20 203960 Uni-ZAPXR 74 1205 1 1205 216 216 268 1 18 Apr. 26, 1999 65 HFPCZ55 203960Uni-ZAP XR 75 2735 341 2735 676 676 269 1 24 25 44 Apr. 26, 1999 66HFPDR62 203960 Uni-ZAP XR 76 2644 1 2644 414 414 270 1 28 29 35 Apr. 26,1999 67 HFPDS07 203960 Uni-ZAP XR 77 3115 2302 3114 2546 2546 271 1 2324 25 Apr. 26, 1999 68 HFVHW43 203960 pBluescript 78 1233 1 1233 92 92272 1 30 31 39 Apr. 26, 1999 69 HFXAV37 203960 Lambda ZAP 79 1520 401520 163 273 1 13 14 36 Apr. 26, 1999 II 70 HFXFZ46 203960 Lambda ZAP 801378 1 1378 258 258 274 1 6 Apr. 26, 1999 II 71 HGBHP91 203960 Uni-ZAPXR 81 1054 1 1054 50 275 1 14 15 52 Apr. 26, 1999 72 HHEAK45 203960pCMVSport 82 2014 87 1935 813 276 1 3 Apr. 26, 1999 3.0 73 HHEOW19PTA-793 pCMVSport 83 1589 1 1589 183 183 277 1 18 19 64 Sep. 27, 19993.0 74 HHFFS40 203960 Uni-ZAP XR 84 1816 1 1816 37 37 278 1 18 19 47Apr. 26, 1999 75 HHGCS78 203960 Lambda ZAP 85 575 46 575 290 290 279 117 18 24 Apr. 26, 1999 II 76 HHPSA85 203960 pBluescript 86 1147 1 1147157 157 280 1 28 29 38 Apr. 26, 1999 77 HHSBI06 203959 Uni-ZAP XR 871049 27 803 690 281 1 5 Apr. 26, 1999 78 HHSBI65 203917 Uni-ZAP XR 881444 1 1431 62 62 282 1 17 18 55 Apr. 8, 1999 79 HHSGL28 203960 Uni-ZAPXR 89 1093 1 1093 453 453 283 1 6 Apr. 26, 1999 80 HISAT67 203959pSport1 90 2154 1061 2142 1239 1239 284 1 35 36 56 Apr. 26, 1999 81HJBCU75 203957 pBluescript 91 1009 1 1009 61 61 285 1 5 Apr. 26, 1999SK- 82 HJMAA03 203957 pCMVSport 92 665 1 665 527 286 1 9 Apr. 26, 19993.0 83 HKABU43 203959 pCMVSport 93 1919 581 1919 755 755 287 1 20 21 281Apr. 26, 1999 2.0 84 HKACI79 PTA-181 pCMVSport 94 1181 1 1181 207 207288 1 14 15 50 Jun. 7, 1999 2.0 85 HKIXC44 203957 pBluescript 95 788 343750 572 572 289 1 26 27 36 Apr. 26, 1999 86 HKTAB41 203957 Uni-ZAP XR 96797 1 797 172 172 290 1 10 Apr. 26, 1999 87 HLDQU79 203959 pCMVSport 971488 1 1488 99 99 291 1 23 24 348 Apr. 26, 1999 3.0 87 HLDQU79 203959pCMVSport 191 3179 163 1474 75 75 385 1 29 30 348 Apr. 26, 1999 3.0 88HLHAP05 203957 Uni-ZAP XR 98 1842 12 1842 45 45 292 1 14 Apr. 26, 199989 HLHCS23 203957 Uni-ZAP XR 99 1427 1 1427 25 25 293 1 24 25 34 Apr.26, 1999 90 HLICE88 203957 pCMVSport 1 100 840 401 824 708 294 1 2 Apr.26, 1999 91 HLMGP50 203957 Lambda ZAP 101 1063 1 1063 214 214 295 1 10Apr. 26, 1999 II 92 HLMMX62 203957 Lambda ZAP 102 268 1 268 185 185 2961 17 18 28 Apr. 26, 1999 II 93 HLQCX36 203957 Lambda ZAP 103 1243 1 124389 89 297 1 16 17 52 Apr. 26, 1999 II 94 HLWAF06 203957 pCMVSport 1042564 1 2564 192 192 298 1 18 19 30 Apr. 26, 1999 3.0 95 HLWDB73 203957pCMVSport 105 1611 1 1611 95 95 299 1 27 28 35 Apr. 26, 1999 3.0 96HLYDF73 203957 pSport1 106 626 1 626 363 300 1 11 12 23 Apr. 26, 1999 97HLYGB19 203959 pSport1 107 2967 1527 2966 1863 1863 301 1 14 Apr. 26,1999 98 HLYGY91 203957 pSport1 108 640 1 640 211 211 302 1 20 21 42 Apr.26, 1999 99 HMDAB29 203957 Uni-ZAP XR 109 1190 1 1190 97 97 303 1 17 1826 Apr. 26, 1999 100 HMDAD44 203957 Uni-ZAP XR 110 1204 1 1204 135 135304 1 8 Apr. 26, 1999 101 HMEDI90 203957 Lambda ZAP 111 2276 362 2219622 305 1 12 13 17 Apr. 26, 1999 II 102 HMIBF07 203957 Uni-ZAP XR 1121738 1 1738 229 229 306 1 6 Apr. 26, 1999 103 HMICP65 203979 Uni-ZAP XR113 2048 1 2048 249 249 307 1 16 17 30 Apr. 29, 1999 104 HMSHC86 203957Uni-ZAP XR 114 1724 1 1724 37 37 308 1 20 21 93 Apr. 26, 1999 105HMUAN45 203918 pCMVSport 115 2709 1 2709 239 239 309 1 25 26 227 Apr. 8,1999 3.0 106 HMVBC31 203957 pSport1 116 2556 1327 2546 1437 1437 310 132 33 40 Apr. 26, 1999 107 HMWBL03 203957 Uni-ZAP XR 117 2596 80 2596137 137 311 1 1 2 397 Apr. 26, 1999 108 HNFGR08 203957 Uni-ZAP XR 1181436 1 1436 314 312 1 17 18 43 Apr. 26, 1999 109 HNGAK51 203957 Uni-ZAPXR 119 915 1 915 248 248 313 1 23 24 32 Apr. 26, 1999 110 HNGDX18PTA-181 Uni-ZAP XR 120 1425 1 1425 237 237 314 1 30 31 243 Jun. 7, 1999110 HNGDX18 PTA-181 Uni-ZAP XR 192 1411 1 1411 231 231 386 1 18 19 132Jun. 7, 1999 111 HNGGP65 203957 Uni-ZAP XR 121 541 1 541 181 181 315 115 16 68 Apr. 26, 1999 112 HNGIV64 203957 Uni-ZAP XR 122 1047 1 1047 221316 1 8 Apr. 26, 1999 113 HNGMW45 203959 Uni-ZAP XR 123 1530 1 1530 452452 317 1 26 27 43 Apr. 26, 1999 114 HNGPJ25 203959 Uni-ZAP XR 124 853129 853 544 544 318 1 20 21 25 Apr. 26, 1999 115 HNHEN82 203918 Uni-ZAPXR 125 573 1 573 78 319 1 13 14 17 Apr. 8, 1999 116 HNHFE71 203959Uni-ZAP XR 126 903 1 903 598 598 320 1 21 Apr. 26, 1999 117 HNHKV56203959 Uni-ZAP XR 127 1653 1 1653 294 294 321 1 31 32 66 Apr. 26, 1999118 HOACG07 203959 Uni-ZAP XR 128 1298 772 1249 778 778 322 1 21 22 123Apr. 26, 1999 119 HODBB70 203918 Uni-ZAP XR 129 604 1 604 173 323 1 7 827 Apr. 8, 1999 120 HOEBK60 203959 Uni-ZAP XR 130 2218 1449 2216 17141714 324 1 39 40 43 Apr. 26, 1999 121 HOFAA78 203959 pSport1 131 1356 11356 48 325 1 25 26 71 Apr. 26, 1999 122 HOFNB74 203959 pCMVSport 1321036 1 1036 138 138 326 1 24 25 39 Apr. 26, 1999 2.0 123 HORBS82 203959Uni-ZAP XR 133 1125 1 1125 21 327 1 19 20 39 Apr. 26, 1999 124 HOSDO75PTA-181 Uni-ZAP XR 134 902 1 902 88 88 328 1 28 Jun. 7, 1999 125 HOUDR07203959 Uni-ZAP XR 135 1911 1 1911 170 170 329 1 27 28 65 Apr. 26, 1999126 HPEAD23 203959 Uni-ZAP XR 136 582 1 582 188 188 330 1 13 14 93 Apr.26, 1999 127 HPFCI36 PTA-181 Uni-ZAP XR 137 879 1 879 94 94 331 1 17 1819 Jun. 7, 1999 128 HPFDI37 PTA-181 Uni-ZAP XR 138 352 1 352 38 38 332 117 Jun. 7, 1999 129 HPIAA80 203959 Uni-ZAP XR 139 919 312 919 314 333 113 14 37 Apr. 26, 1999 130 HPJCW58 203918 Uni-ZAP XR 140 1165 1 1165 177177 334 1 19 20 28 Apr. 8, 1999 131 HPRBH85 203959 Uni-ZAP XR 141 1673558 1648 684 684 335 1 18 19 134 Apr. 26, 1999 132 HPRCA64 203959Uni-ZAP XR 142 2805 1701 2757 1810 1810 336 1 22 23 39 Apr. 26, 1999 133HPRCD35 PTA-181 Uni-ZAP XR 143 709 1 689 265 337 1 16 17 35 Jun. 7, 1999134 HPTRM02 203959 pBluescript 144 1760 658 1680 885 885 338 1 16 17 80Apr. 26, 1999 135 HRAAD30 PTA-181 pCMVSport 145 1496 1 1496 220 339 1 1920 25 Jun. 7, 1999 3.0 136 HRADF49 PTA-181 pCMVSport 146 2704 1 2684 169169 340 1 39 40 253 Jun. 7, 1999 3.0 137 HRDDQ39 203959 Uni-ZAP XR 147776 1 773 215 341 1 17 18 46 Apr. 26, 1999 138 HRDEX93 203959 Uni-ZAP XR148 1681 711 1638 649 649 342 1 20 21 72 Apr. 26, 1999 139 HROEA08PTA-181 Uni-ZAP XR 149 281 1 281 50 50 343 1 25 26 33 Jun. 7, 1999 140HRTAP63 203979 pBluescript 150 2576 891 2576 959 959 344 1 28 29 42 Apr.29, 1999 SK- 141 HSAVW42 203959 Uni-ZAP XR 151 595 1 595 129 129 345 116 17 22 Apr. 26, 1999 142 HSAYC41 203959 Uni-ZAP XR 152 214 1 214 106106 346 1 16 17 36 Apr. 26, 1999 143 HSLHX15 203959 Uni-ZAP XR 153 655 1655 485 485 347 1 20 21 41 Apr. 26, 1999 144 HSNBM34 203959 Uni-ZAP XR154 2186 1391 1765 1508 348 1 14 15 62 Apr. 26, 1999 145 HSSEF77 203959Uni-ZAP XR 155 1053 1 1053 184 349 1 25 26 60 Apr. 26, 1999 146 HT4FV41PTA-181 Uni-ZAP XR 156 1764 1 1764 39 350 1 16 17 137 Jun. 7, 1999 147HT5FX79 203959 Uni-ZAP XR 157 682 59 682 228 351 1 17 18 50 Apr. 26,1999 148 HT5GR59 203959 Uni-ZAP XR 158 1743 1 1743 135 135 352 1 23 2431 Apr. 26, 1999 149 HTAEI78 203918 Uni-ZAP XR 159 1623 1 1623 632 632353 1 4 Apr. 8, 1999 150 HTEAG62 203959 Uni-ZAP XR 160 2221 57 2221 10171017 354 1 20 21 22 Apr. 26, 1999 151 HTEDJ28 203959 Uni-ZAP XR 161 12471 1247 287 355 1 18 19 45 Apr. 26, 1999 152 HTEDS12 203918 Uni-ZAP XR162 1587 1 1587 260 260 356 1 24 25 36 Apr. 8, 1999 153 HTEHA56 203959Uni-ZAP XR 163 1402 529 1400 280 357 1 5 6 88 Apr. 26, 1999 154 HTEJD29203959 Uni-ZAP XR 164 1324 1 1324 101 101 358 1 23 Apr. 26, 1999 155HTENR63 PTA-792 Uni-ZAP XR 165 1591 1 1591 132 132 359 1 20 21 56 Sep.27, 1999 156 HTGGM44 203959 Uni-ZAP XR 166 3016 1 2761 179 179 360 1 1819 84 Apr. 26, 1999 157 HTHBZ06 203959 Uni-ZAP XR 167 623 193 619 318318 361 1 1 Apr. 26, 1999 158 HTLBT80 203959 Uni-ZAP XR 168 2101 8171881 912 912 362 1 27 28 129 Apr. 26, 1999 159 HTLDU78 203918 Uni-ZAP XR169 1318 1 1318 219 219 363 1 8 Apr. 8, 1999 160 HTLFA13 203918 Uni-ZAPXR 170 1160 1 1160 209 364 1 8 9 31 Apr. 8, 1999 161 HTLGI89 203959Uni-ZAP XR 171 2377 1205 2377 1802 1802 365 1 16 17 37 Apr. 26, 1999 162HTNBK13 203959 pBluescript 172 1160 295 1148 534 534 366 1 16 17 21 Apr.26, 1999 SK- 163 HTOAM11 203918 Uni-ZAP XR 173 1200 1 1200 89 89 367 124 25 34 Apr. 8, 1999 164 HTOHO21 203918 Uni-ZAP XR 174 727 1 727 439368 1 5 6 63 Apr. 8, 1999 165 HTPCO75 PTA-181 Uni-ZAP XR 175 1467 1 146773 369 1 23 24 40 Jun. 7, 1999 166 HTSFJ32 203918 pBluescript 176 1257517 1257 93 93 370 1 18 Apr. 8, 1999 167 HTTCB60 PTA-181 Uni-ZAP XR 1771504 1 1504 84 84 371 1 17 18 266 Jun. 7, 1999 168 HTTEE41 203959Uni-ZAP XR 178 1973 864 1968 1171 372 1 8 Apr. 26, 1999 169 HTWEH94203918 pSport1 179 1361 1 1361 66 66 373 1 43 44 81 Apr. 8, 1999 170HTXFA72 PTA-181 Uni-ZAP XR 180 1861 1 1861 192 192 374 1 17 18 29 Jun.7, 1999 171 HTXKF95 203959 Uni-ZAP XR 181 884 79 875 330 330 375 1 28 2978 Apr. 26, 1999 172 HUKDF20 203918 Lambda ZAP 182 1105 1 1105 214 214376 1 20 21 33 Apr. 8, 1999 II 173 HUSCJ14 PTA- Lambda ZAP 183 3342 13342 74 74 377 1 30 31 196 1838 II May 9, 2000 174 HUVDJ48 203918Uni-ZAP XR 184 1827 1 1827 196 196 378 1 5 Apr. 8, 1999 175 HBDAB91203917 pSport1 185 1007 320 1007 671 671 379 1 19 20 29 Apr. 8, 1999 175HBDAB91 203917 pSport1 193 687 1 687 351 351 387 1 19 20 29 Apr. 8, 1999176 HELGG84 203960 Uni-ZAP XR 186 1109 12 1109 147 147 380 1 16 17 22Apr. 26, 1999 176 HELGG84 203960 Uni-ZAP XR 194 1109 12 1109 147 147 3881 16 17 22 Apr. 26, 1999 177 HILCA24 203960 pBluescript 187 1982 1531982 191 191 381 1 29 30 327 Apr. 26, 1999 SK- 177 HILCA24 203960pBluescript 195 1980 151 1976 189 189 389 1 29 30 327 Apr. 26, 1999 SK-178 HYABC84 203959 pCMVSport 188 1478 833 1306 1080 1080 382 1 28 29 62Apr. 26, 1999 3.0 178 HYABC84 203959 pCMVSport 196 1338 768 1238 10151015 390 1 28 29 62 Apr. 26, 1999 3.0 179 HMCAZ04 203917 Uni-ZAP XR 1891301 1 1301 97 97 383 1 20 21 35 Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAPXR 197 1735 407 1671 498 498 391 1 20 21 35 Apr. 8, 1999 179 HMCAZ04203917 Uni-ZAP XR 198 1733 406 1670 106 106 392 1 25 26 450 Apr. 8, 1999179 HMCAZ04 203917 Uni-ZAP XR 199 1733 405 1670 497 497 393 1 20 21 35Apr. 8, 1999 179 HMCAZ04 203917 Uni-ZAP XR 200 1733 405 1670 106 106 3941 25 26 450 Apr. 8, 1999 180 HE8FD92 203979 Uni-ZAP XR 190 3977 19863960 2141 2141 384 1 25 26 43 Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAPXR 201 1995 1 1978 157 157 395 1 25 26 43 Apr. 29, 1999 180 HE8FD92203979 Uni-ZAP XR 202 4102 2114 4085 2268 2268 396 1 25 26 43 Apr. 29,1999 180 HE8FD92 203979 Uni-ZAP XR 203 4907 2918 4890 2 397 1 1 2 471Apr. 29, 1999 180 HE8FD92 203979 Uni-ZAP XR 204 2908 918 2891 1074 1074398 1 25 26 43 Apr. 29, 1999Table 1B (Comprised of Tables 1B.1 and 1B.2)

The first column in Table 1B.1 and Table 1B.2 provides the gene numberin the application corresponding to the clone identifier. The secondcolumn in Table 1B.1 and Table 1B.2 provides a unique “Clone ID:” forthe cDNA clone related to each contig sequence disclosed in Table 1B.1and Table 1B.2. This clone ID references the cDNA clone which containsat least the 5′ most sequence of the assembled contig and at least aportion of SEQ ID NO:X as determined by directly sequencing thereferenced clone. The referenced clone may have more sequence thandescribed in the sequence listing or the clone may have less. In thevast majority of cases, however, the clone is believed to encode afull-length polypeptide. In the case where a clone is not full-length, afull-length cDNA can be obtained by methods described elsewhere herein.The third column in Table 1B.1 and Table 1B.2 provides a unique “ContigID” identification for each contig sequence. The fourth column in Table1B.1 and Table 1B.2 provides the “SEQ ID NO:” identifier for each of thecontig polynucleotide sequences disclosed in Table 1B.

Table 1B.1

The fifth column in Table 1B.1, “ORF (From-To)”, provides the location(i.e., nucleotide position numbers) within the polynucleotide sequence“SEQ ID NO:X” that delineate the preferred open reading frame (ORF)shown in the sequence listing and referenced in Table 1B.1, column 6, asSEQ ID NO:Y. Where the nucleotide position number “To” is lower than thenucleotide position number “From”, the preferred ORF is the reversecomplement of the referenced polynucleotide sequence. The sixth columnin Table 1B.1 provides the corresponding SEQ ID NO:Y for the polypeptidesequence encoded by the preferred ORF delineated in column 5. In oneembodiment, the invention provides an amino acid sequence comprising, oralternatively consisting of, a polypeptide encoded by the portion of SEQID NO:X delineated by “ORF (From-To)”. Also provided are polynucleotidesencoding such amino acid sequences and the complementary strand thereto.Column 7 in Table 1B.1 lists residues comprising epitopes contained inthe polypeptides encoded by the preferred ORF (SEQ ID NO:Y), aspredicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl.Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performedusing the computer program PROTEAN (Version 3.11 for the PowerMacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Inspecific embodiments, polypeptides of the invention comprise, oralternatively consist of, at least one, two, three, four, five or moreof the predicted epitopes as described in Table 1B. It will beappreciated that depending on the analytical criteria used to predictantigenic determinants, the exact address of the determinant may varyslightly.

Column 8 in Table 1B.1 provides a chromosomal map location for certainpolynucleotides of the invention. Chromosomal location was determined byfinding exact matches to EST and cDNA sequences contained in the NCBI(National Center for Biotechnology Information) UniGene database. Eachsequence in the UniGene database is assigned to a “cluster”; all of theESTs, cDNAs, and STSs in a cluster are believed to be derived from asingle gene. Chromosomal mapping data is often available for one or moresequence(s) in a UniGene cluster; this data (if consistent) is thenapplied to the cluster as a whole. Thus, it is possible to infer thechromosomal location of a new polynucleotide sequence by determining itsidentity with a mapped UniGene cluster.

A modified version of the computer program BLASTN (Altshul, et al., J.Mol. Biol. 215:403-410 (1990), and Gish, and States, Nat. Genet.3:266-272) (1993) was used to search the UniGene database for EST orcDNA sequences that contain exact or near-exact matches to apolynucleotide sequence of the invention (the ‘Query’). A sequence fromthe UniGene database (the ‘Subject’) was said to be an exact match if itcontained a segment of 50 nucleotides in length such that 48 of thosenucleotides were in the same order as found in the Query sequence. Ifall of the matches that met this criteria were in the same UniGenecluster, and mapping data was available for this cluster, it isindicated in Table 1B under the heading “Cytologic Band”. Where acluster had been further localized to a distinct cytologic band, thatband is disclosed; where no banding information was available, but thegene had been localized to a single chromosome, the chromosome isdisclosed.

Once a presumptive chromosomal location was determined for apolynucleotide of the invention, an associated disease locus wasidentified by comparison with a database of diseases which have beenexperimentally associated with genetic loci. The database used was theMorbid Map, derived from OMIM™ and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.) 2000;. If theputative chromosomal location of a polynucleotide of the invention(Query sequence) was associated with a disease in the Morbid Mapdatabase, an OMIM reference identification number was noted in column 9,Table 1B.1, labelled “OEM Disease Reference(s). Table 5 is a key to theOMIM reference identification numbers (column 1), and provides adescription of the associated disease in Column 2.

Table 1B.2

Column 5, in Table 1B.2, provides an expression profile and librarycode:count for each of the contig sequences (SEQ ID NO:X) disclosed inTable 1B, which can routinely be combined with the information providedin Table 4 and used to determine the tissues, cells, and/or cell linelibraries which predominantly express the polynucleotides of theinvention. The first number in Table 1B.2, column 5 (preceding thecolon), represents the tissue/cell source identifier code correspondingto the code and description provided in Table 4. The second number incolumn 5 (following the colon) represents the number of times a sequencecorresponding to the reference polynucleotide sequence was identified inthe corresponding tissue/cell source. Those tissue/cell sourceidentifier codes in which the first two letters are “R” designateinformation generated using DNA array technology. Utilizing thistechnology, cDNAs were amplified by PCR and then transferred, induplicate, onto the array. Gene expression was assayed throughhybridization of first strand cDNA probes to the DNA array. cDNA probeswere generated from total RNA extracted from a variety of differenttissues and cell lines. Probe synthesis was performed in the presence of³³P dCTP, using oligo (dT) to prime reverse transcription. Afterhybridization, high stringency washing conditions were employed toremove non-specific hybrids from the array. The remaining signal,emanating from each gene target, was measured using a Phosphorimager.Gene expression was reported as Phosphor Stimulating Luminescence (PSL)which reflects the level of phosphor signal generated from the probehybridized to each of the gene targets represented on the array. A localbackground signal subtraction was performed before the total signalgenerated from each array was used to normalize gene expression betweenthe different hybridizations. The value presented after “[array code]:”represents the mean of the duplicate values, following backgroundsubtraction and probe normalization. One of skill in the art couldroutinely use this information to identify normal and/or diseasedtissue(s) which show a predominant expression pattern of thecorresponding polynucleotide of the invention or to identifypolynucleotides which show predominant and/or specific tissue and/orcell expression. TABLE 1B.1 SEQ AA ID SEQ OMIM Gene cDNA Clone ContigNO: ORF ID Cytologic Disease No: ID ID: X (From-To) NO: Y PredictedEpitopes Band Reference(s): 1 H6EDM64 841331 11 1448-1468 205 11q13102200, 106100, 131100, 131100, 131100, 133780, 147050, 153700, 161015,164009, 168461, 168461, 168461, 180721, 180840, 191181, 193235, 209901,232600, 259700, 259770, 600045, 600319, 600528, 601884 2 H6EEU40 75704812 175-318 206 11q12.1 106100, 147050, 259700, 259770, 600045, 601884 3HACAB68 584773 13 135-371 207 Leu-6 to Ser-12. 4 HACBJ56 847112 14250-327 208 Arg-14 to Ile-24. 5 HADMB15 847116 15 238-300 209 6 HAGDW20637489 16 238-291 210 7 HAGEQ79 828055 17 515-550 211 8 HAJBV67 86641518 605-1684 212 Arg-24 to Trp-44, 10q23.33 157640, 174900, 236730,600512 Leu-87 to Ser-93, Arg-119 to Trp-125, Pro-206 to Lys-211, Glu-280to Trp-286. 9 HAJCH70 827275 19 284-400 213 10 HAOAG15 852204 20  8-3511 214 Asp-26 to Leu-32, 1q21 104770, 107670, 110700, 135940,145001, Trp-62 to Asp-72, 146790, 152445, 152445, 159001, 174000, Gln-95to His-101, 179755, 182860, 182860, 182860, 191315, Thr-158 to Thr-164,230800, 230800, 266200, 600897, 601105, Phe-222 to Glu-227, 601412,601652, 602491 Asn-234 to Thr-245, Gly-256 to Glu-266, Gly-277 toGlu-283, Arg-310 to Ser-317, Ser-327 to Phe-333, Ser-360 to Ser-366. 11HATCD80 826098 21 296-409 215 12 HATCI03 580805 22 271-324 216 Lys-8 toTrp-13. 13 HBAGD86 838799 23 521-580 217 14 HBHAA05 603174 24 110-286218 15 HBHAA81 846465 25  28-639 219 3p21.32 116806, 168468, 182280,600163 16 HBIAA59 806303 26 1877-2287 220 Arg-34 to Ser-39, Pro-45 toIle-55. 17 HBJAC40 841235 27 329-370 221 16p13.3 141750, 141800, 141800,141800, 141800, 141850, 141850, 141850, 141850, 141850, 156850, 186580,191092, 600140, 600273, 601313, 601785 18 HBJCR46 815649 28  589-2787222 Met-1 to Ala-8, 15q12 103581, 146150, 182279, 203200, 203200, Phe-42to Asp-57, 227220, 601623, 601800, 601889, 602117 Tyr-105 to Thr-110,His-121 to Cys-127, Asp-154 to Lys-181, Arg-186 to Pro-210, Ala-233 toAsp-252, Ser-296 to Ser-306, Pro-313 to Ser-320, Gln-331 to Gly-346,Ser-355 to Thr-360, Cys-386 to Phe-395, Ser-400 to Glu-425, Thr-440 toThr-446, Pro-449 to Cys-466, Glu-470 to Thr-509, Ser-512 to Asp-533,Ala-544 to Arg-550, Arg-562 to Glu-571, Lys-587 to Thr-594, Asp-713 toGlu-733. 19 HBJDW56 520401 29 121-147 223 20 HBJEL16 847030 30 115-225224 1q23.1- 107300, 131210, 136132, 145001, 173610, q23.2 249270, 60165221 HBJIG20 866159 31 321-554 225 22 HBJKD16 853358 32  78-173 226 2p14203800 23 HBMTY48 637521 33 660-944 227 Glu-35 to Pro-50. 24 HBMUH74866160 34 344-430 228 12p11.22 112410, 135700, 168470, 200990 25 HBQAB79810542 35 190-225 229 4q31.1 189800, 600983 26 HBXCX15 637542 36 72-77230 27 HCDCY76 837972 37 860-967 231 Pro-20 to Phe-25. 11q14-q21 133780,203100, 203100, 245000 28 HCEDR26 771144 38 177-344 232 29 HCEEU18688041 39 209-340 233 30 HCEGX05 827060 40 237-284 234 Pro-4 to Phe-11.20q13 600281, 600281 31 HCFLN88 610000 41 101-178 235 7q11.23 116860,129900, 233700, 600079 32 HCHAB84 834326 42 304-747 236 Asn-47 toLeu-52, Tyr-134 to Trp-143. 33 HCMSX51 788643 43 539-781 237 Leu-57 toGlu-66. 8p21 152760, 180100, 185430, 602629 34 HCNCO11 775086 44 101-145238 35 HCNSD29 862314 45 1145-1240 239 2q23.3 36 HCQBH72 637548 46 31-174 240 37 HCQCC96 845066 47 782-919 241 38 HCQCJ56 832157 48728-733 242 39 HCUDD64 835082 49 256-402 243 Met-1 to Ser-6, 19p13.3108725, 120700, 133171, 136836, 145981, Gln-32 to Asn-39. 147141,164953, 188070, 600957, 601238, 601846, 602216, 602477 40 HCWAE64 53589350 410-427 244 41 HDPDJ58 587265 51 279-341 245 42 HDPFU43 790189 52220-378 246 22q12.1 123620, 188826, 600850, 601669 43 HDPGE24 801947 53173-394 247 44 HDPIU94 813352 54 208-279 248 8p21.1 138300, 240400,602629 45 HDPIY31 886159 55 268-375 249 20q13.33 46 HDPOC24 777493 56418-819 250 Pro-36 to Cys-42, 9q34.12 Pro-44 to Cys-54, Arg-100 toGly-105. 47 HDPPD93 637588 57 28-66 251 48 HDPPQ30 684292 58 220-336 25249 HDQHM36 852328 59 129-275 253 50 HDTFX18 801957 60 164-226 254 51HE2CM39 553651 61 10-51 255 52 HE2HC60 753265 62 273-392 256 Thr-26 toGln-31. 1q42.2 106150, 106150, 214500, 600996, 601975, 602759 53 HE2PO93771655 63 770-898 257 3p21.3 116806, 120120, 120120, 120120, 120436,120436, 120436, 138320, 168468, 182280, 600163 54 HE6FU11 827236 64145-825 258 55 HE6FV29 588454 65 210-311 259 56 HE8TY46 899528 661413-1976 260 11p11.2- 133701, 168500, 171650, 176930, 176930, p11.12600623, 600811, 600958 57 HE9EA10 827796 67 212-448 261 Arg-6 to Trp-11.58 HEBCY54 600355 68 172-528 262 Arg-18 to Lys-26, 8p22-p21 148370,152760, 180100, 185430, 238600, Gly-35 to Ala-42, 238600, 238600,238600, 600143, 601385, Gln-61 to Gly-67. 602629 59 HEBFR46 847064 69200-289 263 Met-1 to Thr-6. 60 HEBGE07 798096 70 106-234 264 61 HEGAU15834379 71  59-163 265 62 HETCI16 844543 72 237-359 266 Met-1 to Trp-9.63 HFCEI04 692438 73 136-264 267 Asn-21 to Gly-28. 64 HFCFE20 701985 74216-272 268 10q26 176943, 176943, 176943, 176943, 176943, 258870,263700, 601969, 601969, 602084 65 HFPCZ55 840840 75 676-810 269 11p15108985, 186921, 602092 66 HFPDR62 839400 76 414-521 270 67 HFPDS07821646 77 2546-2623 271 2q32-q34 100690, 100730, 118800, 123660, 135600,142989, 156232, 157655, 178600, 186860, 201460, 205100, 262000, 278250,600258, 601277, 601318 68 HFVHW43 570948 78  92-211 272 69 HFXAV37626595 79 163-273 273 70 HFXFZ46 600361 80 258-278 274 71 HGBHP91 69301181  50-208 275 72 HHEAK45 765278 82 813-824 276 6p21.33 248611 73HHEOW19 886174 83 183-377 277 Ala-41 to Pro-57. 1q42 106150, 106150,145260, 173870, 173870, 600759, 600996, 601744, 601975 74 HHFFS40 82405984  37-180 278 5p14.1 123000 75 HHGCS78 634605 85 290-364 279 17q11.1182138, 600881, 601954 76 HHPSA85 658695 86 157-273 280 77 HHSBI06639097 87 690-707 281 78 HHSBI65 801910 88  62-229 282 Ala-16 to Val-35.8q24.3 188450, 188450, 188450 79 HHSGL28 801912 89 453-473 283 80HISAT67 843549 90 1239-1409 284 2p23.3 176830, 176830, 182601, 229800,602134 81 HJBCU75 638329 91 61-78 285 82 HJMAA03 824062 92 527-556 28683 HKABU43 838573 93  755-1600 287 Ile-69 to Ala-74, Ala-122 to Ser-129,Thr-160 to Glu-170, Lys-197 to Arg-202. 84 HKACI79 853361 94 207-359 288Ser-37 to Gly-43. 85 HKIXC44 716213 95 572-682 289 86 HKTAB41 695732 96172-204 290 87 HLDQU79 740755 97  99-1142 291 Leu-68 to Lys-74, Tyr-109to Lys-115, Gln-200 to Val-205, Lys-207 to Lys-214, Glu-237 to Ile-244,Ala-271 to Thr-279, Ser-317 to Ser-329, Gln-342 to Gly-348. HLDQU79837599 191  75-1121 385 88 HLHAP05 638476 98 45-89 292 Gln-4 to Leu-14.89 HLHCS23 560663 99  25-129 293 90 HLICE88 840321 100 708-716 294 4q28107250, 134820, 134820, 134820, 134830, 134850, 134850, 181600, 189800,266300 91 HLMGP50 647603 101 214-246 295 92 HLMMX62 688051 102 185-268296 Gln-20 to Lys-28. 93 HLQCX36 584786 103  89-247 297 Pro-35 toSer-40. 94 HLWAF06 658701 104 192-284 298 95 HLWDB73 838453 105  95-202299 1q32.2 145260, 600759, 601975 96 HLYDF73 566869 106 363-434 300 97HLYGB19 838083 107 1863-1907 301 2p23.3 176830, 176830, 182601, 229800,602134 98 HLYGY91 658703 108 211-339 302 99 HMDAB29 584789 109  97-177303 100 HMDAD44 566854 110 135-161 304 101 HMEDI90 840077 111 622-675305 Ser-7 to Thr-13. 102 HMIBF07 603528 112 229-249 306 103 HMICP65847403 113 249-341 307 104 HMSHC86 840402 114  37-318 308 Arg-32 toGln-37, Arg-68 to Phe-73. 105 HMUAN45 833072 115 239-922 309 Pro-33 toGly-45, 11q13.5 133780, 266150, 276903, 276903, 276903 Cys-121 toGly-131, Ala-155 to His-166, Gly-180 to Gln-185. 106 HMVBC31 825598 1161437-1559 310 Ser-33 to Tyr-39. 1p36.21 120550, 120570, 120575, 153454,256700 107 HMWBL03 822861 117  137-1327 311 Met-1 to Leu-11, Val-13 toLys-19, Thr-30 to Asp-39, Thr-49 to Gly-68, Ala-78 to Gly-111, Pro-140to Thr-163, Ser-169 to Ser-185, Glu-197 to Lys-204, Lys-210 to Asp-215,Glu-220 to Ser-231, Ser-255 to Leu-266, Thr-269 to Asp-288, Cys-300 toVal-309, Phe-331 to Cys-339, Ser-362 to Ile-373. 108 HNFGR08 825417 118314-445 312 109 HNGAK51 603910 119 248-346 313 110 HNGDX18 1145071 120237-965 314 Ser-21 to Ser-39, Gln-45 to Gln-61, Cys-124 to Ser-139.HNGDX18 866177 192 231-629 386 Ser-21 to Ser-39, Gln-45 to Gln-61,Cys-124 to Gly-130. 111 HNGGP65 597449 121 181-387 315 112 HNGIV64561572 122 221-247 316 113 HNGMW45 838613 123 452-583 317 114 HNGPJ25834942 124 544-621 318 115 HNHEN82 836157 125  78-131 319 116 HNHFE71834487 126 598-663 320 117 HNHKV56 800877 127 294-494 321 118 HOACG07792928 128  778-1149 322 Pro-32 to Ser-42, 20p13 192340, 234200 Cys-51to Gly-83, Gly-87 to Ser-93. 119 HODBB70 520196 129 173-256 323 120HOEBK60 789396 130 1714-1845 324 Lys-5 to Thr-10, Gln-36 to Gly-43. 121HOFAA78 836646 131  48-263 325 Trp-1 to Arg-7, 19q13.33 134790, 600040Pro-65 to Gly-70. 122 HOFNB74 762821 132 138-257 326 Ser-30 to Ser-36.12q12- 181430, 600194, 600231, 600808, 601284, 12q14.3 601769, 601769,602116 123 HORBS82 638293 133  21-140 327 Gly-30 to Ser-35. 124 HOSDO75862049 134  88-174 328 Phe-2 to Ser-8, 11q13.4 133780, 266150 Phe-21 toSer-26. 125 HOUDR07 745404 135 170-367 329 Pro-27 to Arg-34. 19p13.3108725, 120700, 133171, 136836, 145981, 147141, 164953, 188070, 600957,601238, 601846, 602216, 602477 126 HPEAD23 773409 136 188-469 330 Ala-54to Lys-59. 127 HPFCI36 855966 137  94-153 331 10q23.31 157640, 174900,236730, 600512 128 HPFDI37 862056 138 38-91 332 22q11.21 123620, 151410,600850 129 HPIAA80 829972 139 314-427 333 130 HPJCW58 612866 140 177-263334 Leu-16 to Gly-21. 131 HPRBH85 695752 141  684-1088 335 Glu-121 toLeu-126. 3q21.1 106165, 117700, 117700, 150210, 169600, 180380, 180380,180380, 203500, 232050, 276902, 600882, 601199, 601199, 601199, 601471,601682 132 HPRCA64 824074 142 1810-1929 336 2q32 100690, 142989, 156232,178600, 278250, 600258 133 HPRCD35 853551 143 265-372 337 Asp-16 toGln-27. 134 HPTRM02 812879 144  885-1127 338 His-48 to Ser-61, 7 Ala-66to Val-72. 135 HRAAD30 866187 145 220-297 339 2p23.3 176830, 176830,182601, 229800, 602134 136 HRADF49 866481 146 169-930 340 Pro-85 toAsp-99, 2q36.1 120070, 120131, 120131, 138030, 259900 Arg-163 toArg-170, Gln-183 to Thr-189, Pro-201 to Ser-209, Ser-216 to Gly-222. 137HRDDQ39 840405 147 215-355 341 Gly-27 to Pro-35. 138 HRDEX93 816046 148649-867 342 1p34 130500, 133200, 138140, 168360, 171760, 171760, 176100,176100, 178300, 230000, 255800 139 HROEA08 866190 149  50-151 343 2q31.1100690, 142989, 156232, 178600, 600258 140 HRTAP63 780698 150  959-1087344 Asn-2 to Trp-13. 2p23.3 176830, 176830, 182601, 229800, 602134 141HSAVW42 637660 151 129-197 345 3p22.3 182280, 227646, 261510, 600163,601154 142 HSAYC41 688057 152 106-213 346 Lys-23 to Lys-36. 11q12.1106100, 147050, 259700, 259770, 600045, 601884 143 HSLHX15 777861 153485-610 347 Arg-28 to Arg-35. 144 HSNBM34 635131 154 1508-1696 348Ala-17 to Thr-26, 17p13-p11 100710, 138190, 254210, 271900, 600179,Gly-49 to Gln-62. 600977, 601202, 601777 145 HSSEF77 658725 155 184-366349 Arg-22 to Lys-27, 2p12 147200, 178640, 216900 Leu-30 to Asn-39. 146HT4FV41 853400 156  39-452 350 Ala-15 to Gln-22, 19p13.3 108725, 120700,133171, 136836, 145981, Gly-36 to Gly-41, 147141, 164953, 188070,600957, 601238, Arg-47 to Pro-63, 601846, 602216, 602477 Pro-85 toHis-98. 147 HT5FX79 794169 157 228-380 351 Glu-1 to Ser-9, Ser-23 toSer-35. 148 HT5GR59 801930 158 135-230 352 8p21.3 602629 149 HTAEI78637684 159 632-646 353 8p21.1 138300, 240400, 602629 150 HTEAG62 812332160 1017-1085 354 151 HTEDJ28 762845 161 287-424 355 Thr-34 to Leu-41.8p22-q11 148370, 180100, 238600, 238600, 238600, 238600, 600143, 601385,602629 152 HTEDS12 838621 162 260-370 356 Ala-29 to Thr-36. 17q21.33109270, 109270, 109270, 109270, 109270, 120150, 120150, 120150, 148065,148080, 154275, 171190, 185800, 221820, 249000, 253250, 600119, 600119,600525, 601844 153 HTEHA56 806461 163 280-546 357 His-10 to Ala-20.19q13.43 154 HTEJD29 695798 164 101-172 358 155 HTENR63 877952 165132-302 359 Pro-22 to Lys-28. 4q24 157147, 248510 156 HTGGM44 842856 166179-433 360 3q23 106165, 110100, 117700, 117700, 150210, 169600, 180380,180380, 180380, 203500, 276902, 601199, 601199, 601199, 601682 157HTHBZ06 832477 167 318-323 361 12q24.31 181405 158 HTLBT80 840045 168 912-1301 362 Ser-107 to Ser-116. 20q11.21- 102700, 102700, 602025q13.11 159 HTLDU78 637702 169 219-245 363 160 HTLFA13 535937 170 209-304364 161 HTLGI89 835069 171 1802-1915 365 162 HTNBK13 831967 172 534-599366 22q12 123620, 133450, 133450, 600850, 601669 163 HTOAM11 664508 173 89-193 367 164 HTOHO21 732808 174 439-630 368 Ile-35 to Cys-42.15q24-q25 118485, 231680, 272800, 272800, 272800, 276700, 602685 165HTPCO75 853645 175  73-195 369 166 HTSFJ32 637720 176  93-149 370 Leu-12to Cys-18. 17p13.1 191170, 191170 167 HTTCB60 853401 177  84-884 371Ser-83 to Asp-88, Val-166 to Gly-181, Pro-193 to Ala-199, Glu-235 toGln-250. 168 HTTEE41 840950 178 1171-1197 372 12q15 181430, 600698,600698, 600698, 600698, 600808, 602116 169 HTWEH94 561680 179  66-311373 170 HTXFA72 853410 180 192-281 374 171 HTXKF95 834438 181 330-566375 Met-1 to Pro-6, Gly-73 to Thr-78. 172 HUKDF20 566823 182 214-315 376173 HUSCJ14 894699 183  74-661 377 Phe-166 to Arg-174, Ser-191 toTyr-196. 174 HUVDJ48 564853 184 196-213 378 175 HBDAB91 864374 185671-760 379 Lys-21 to Gln-29. HBDAB91 789532 193 351-440 387 Lys-21 toGln-29. 176 HELGG84 851137 186 147-215 380 HELGG84 674456 194 147-215388 177 HILCA24 869856 187  191-1174 381 Gln-52 to Arg-57, 5p15.2123000, 602568 Glu-74 to Leu-84, Val-104 to Asp-110, Gly-157 to Gly-163,Asn-185 to Ser-195, Arg-245 to Asp-250, Pro-302 to Pro-310, Thr-316 toTyr-322. HILCA24 782450 195  189-1172 389 Gln-52 to Arg-57, Glu-74 toLeu-84, Val-104 to Asp-110, Gly-157 to Gly-163, Asn-185 to Ser-195,Arg-245 to Asp-250, Pro-302 to Pro-310, Thr-316 to Tyr-322. 178 HYABC84865064 188 1080-1268 382 Pro-3 to Ala-8. 20q11.22 HYABC84 789854 1961015-1203 390 Pro-3 to Ala-8. 179 HMCAZ04 668249 189  97-204 383 Met-1to Pro-7. 15q15 177070, 177070, 182500, 218000, 227220, 243500, 600839,601800 HMCAZ04 887445 197 498-605 391 Met-1 to Pro-7. HMCAZ04 867910 198 106-1455 392 Pro-76 to Phe-81, Gln-95 to Pro-102, Leu-121 to Ile-128,Asp-131 to Ser-137, Thr-174 to Trp-179, Arg-217 to Lys-224, Val-257 toAsn-262, Asn-277 to Glu-283, His-325 to Asn-330, Lys-365 to Thr-377,Pro-404 to Arg-411. HMCAZ04 858210 199 497-604 393 Met-1 to Pro-7.HMCAZ04 839783 200  106-1455 394 Pro-76 to Phe-81, Gln-95 to Pro-102,Leu-121 to Ile-128, Asp-131 to Ser-137, Thr-174 to Trp-179, Arg-217 toLys-224, Val-257 to Asn-262, Asn-277 to Glu-283, His-325 to Asn-330,Lys-365 to Thr-377, Pro-404 to Arg-411. 180 HE8FD92 901142 190 2141-2272384 HE8FD92 888274 201 157-288 395 HE8FD92 869847 202 2268-2399 396HE8FD92 856544 203   2-1414 397 Asp-11 to Tyr-16. HE8FD92 843825 2041074-1205 398

TABLE 1B.2 SEQ ID Tissue Distribution Library Code: Count Gene No: cDNAClone ID Contig ID: NO: X (see Table 4 for Library Codes) 1 H6EDM64841331 11 AR277:22, AR060:22, AR055:21, AR283:18, AR282:18, AR104:16,AR185:16, AR089:16, AR299:16, AR219:14, AR240:14, AR316:13, AR096:12,AR218:12, AR039:11, AR300:11, AR313:11 H0333:6, H0556:5, H0255:5,H0618:4, L0783:4, S0358:3, H0549:3, S0222:3, H0318:3, H0052:3, H0553:3,H0135:3, L0769:3, L3905:3, H0547:3, H0521:3, H0555:3, H0423:3, H0716:2,H0341:2, H0402:2, H0592:2, H0253:2, 50474:2, H0620:2, H0181:2, H0617:2,H0059:2, L0761:2, L0764:2, L0809:2, L5622:2, H0520:2, H0682:2, S0330:2,H0436:2, L0751:2, L0747:2, L0750:2, L0755:2, S0436:2, L0596:2, L0601:2,H0624:1, H0686:1, H0295:1, T0049:1, H0657:1, H0656:1, H0484:1, H0483:1,S0356:1, S0442:1, S0354:1, S0360:1, S0410:1, H0729:1, H0742:1, S0045:1,S0476:1, H0619:1, S0300:1, L0717:1, S0220:1, H0370:1, H0455:1, H0586:1,H0587:1, H0559:1, L0623:1, T0082:1, H0581:1, H0183:1, H0205:1, H0327:1,H0050:1, H0687:1, H0615:1, T0006:1, H0424:1, H0213:1, H0606:1, H0166:1,S0366:1, H0090:1, H0087:1, H0264:1, H0488:1, H0413:1, H0100:1, H0625:1,H0561:1, H0130:1, H0633:1, H0647:1, S0426:1, H0529:1, L0371:1, L0796:1,L0637:1, L5566:1, L0648:1, L0364:1, L0649:1, L0774:1, L0375:1, L0378:1,L0659:1, L0636:1, L5623:1, L4501:1, L0663:1, H0693:1, H0593:1, S0126:1,H0522:1, S0027:1, S0028:1, L0740:1, L0780:1, L0758:1, H0445:1, S0011:1,H0136:1, S0196:1 and H0352:1. 2 H6EEU40 757048 12 AR277:63, AR283:52,AR219:45, AR282:44, AR104:43, AR218:41, AR316:39, AR089:37, AR313:37,AR299:35, AR055:33, AR240:33, AR096:30, AR185:29, AR300:28, AR039:27,AR060:27 L0741:8, H0677:7, L0439:6, H0052:5, H0494:5, L0747:5, S0007:4,H0543:4, H0009:3, L0771:3, L0775:3, L0663:3, L0665:3, L0438:3, H0547:3,H0521:3, H0436:3, L0742:3, L0748:3, L0751:3, S0436:3, H0556:2, H0255:2,S0420:2, S0358:2, S0046:2, L0717:2, S0222:2, H0333:2, H0559:2, H0318:2,H0581:2, H0545:2, H0620:2, H0024:2, H0266:2, H0617:2, H0529:2, L0662:2,L0653:2, L0659:2, L0809:2, L0664:2, H0690:2, H0555:2, L0743:2, L0755:2,L0757:2, L0759:2, L0588:2, H0352:2, H0624:1, L0685:1, L0740:1, H0295:1,S0134:1, H0583:1, H0656:1, H0341:1, S0212:1, H0484:1, L3659:1, S0418:1,S0356:1, S0442:1, S0410:1, H0729:1, H0735:1, H0339:1, H0619:1, S0278:1,H0257:1, H0069:1, H0744:1, H0327:1, H0023:1, S0051:1, T0010:1, H0031:1,H0181:1, H0032:1, H0169:1, S0364:1, H0135:1, H0163:1, H0090:1, H0063:1,H0087:1, H0551:1, H0264:1, H0488:1, H0623:1, H0100:1, S0438:1, S0440:1,L0369:1, L0770:1, L0769:1, L5565:1, L0761:1, L0667:1, L0772:1, L0641:1,L0644:1, L0764:1, L0773:1, L0363:1, L0768:1, L0794:1, L0766:1, L0381:1,L0803:1, L0774:1, L0375:1, L0806:1, L0512:1, L0517:1, L0666:1, H0144:1,H0702:1, S0148:1, L0352:1, H0519:1, L0593:1, H0435:1, H0658:1, H0539:1,S0406:1, H0478:1, H0631:1, S0028:1, S0206:1, L0744:1, L0740:1, L0745:1,L0749:1, L0750:1, L0758:1, H0445:1, S0434:1, L0594:1, S0194:1, H0542:1and H0423:1. 3 HACAB68 584773 13 L0748:4, H0457:3 and S6022:1. 4 HACBJ56847112 14 AR251:7, AR310:6, AR265:6, AR053:6, AR060:6, AR182:6, AR055:6,AR312:5, AR309:5, AR273:5, AR282:5, AR061:5, AR206:5, AR241:5, AR194:5,AR186:5, AR270:4, AR213:4, AR052:4, AR266:4, AR218:4, AR089:4, AR274:4,AR253:4, AR269:4, AR291:4, AR248:4, AR296:4, AR183:4, AR240:4, AR104:4,AR293:4, AR205:4, AR284:4, AR289:3, AR313:3, AR184:3, AR299:3, AR247:3,AR185:3, AR316:3, AR231:3, AR298:3, AR033:3, AR243:3, AR268:3, AR290:3,AR283:3, AR295:3, AR300:3, AR246:3, AR277:3, AR232:3, AR175:3, AR096:3,AR219:3, AR177:3, AR234:3, AR233:3, AR275:3, AR237:3, AR238:3, AR285:3,AR227:3, AR202:3, AR263:3, AR292:3, AR204:2, AR286:2, AR314:2, AR267:2,AR280:2, AR229:2, AR258:2, AR039:2, AR294:2, AR256:2, AR259:2, AR281:1,AR315:1, AR226:1, AR244:1, AR249:1 H0661:1, S0045:1, H0550:1, S0280:1,S0010:1, H0028:1, L0764:1, L0803:1, L0805:1, L0665:1, S0053:1, H0670:1,L0748:1, L0731:1 and L0581:1. 5 HADMB15 847116 15 AR104:19, AR218:19,AR219:16, AR089:11, AR313:8, AR055:8, AR060:7, AR299:6, AR282:5,AR300:5, AR039:5, AR240:5, AR316:5, AR185:5, AR277:4, AR283:4, AR096:3L0595:2, L0442:1, L0005:1, L3653:1, H0390:1, H0081:1, H00241, L0770:1,L5566:1, L0651:1, L0565:1, L0439:1, L0747:1, L0752:1, H0445:1, L0592:1and L0599:1. 6 HAGDW20 637489 16 AR313:22, AR039:22, AR277:20, AR299:17,AR089:17, AR300:14, AR185:14, AR218:13, AR096:13, AR240:13, AR219:13,AR104:13, AR316:13, AR282:12, AR283:11, AR060:11, AR055:11 S0010:1 andH0616:1. 7 HAGEQ79 828055 17 AR104:37, AR283:37, AR277:26, AR055:20,AR185:20, AR316:18, AR299:17, AR282:16, AR313:16, AR219:16, AR240:16,AR089:16, AR218:14, AR060:14, AR096:13, AR039:12, AR300:10 L0805:6,L0809:4, L0803:3, L0779:3, L0794:2, L0776:2, L0438:2, L0439:2, L0745:2,L0747:2, S0436:2, S0408:1, T0082:1, S0010:1, H0052:1, T0010:1, H0598:1,L0770:1, L0774:1, L0783:1, L0788:1, L0665:1, L0742:1, L0777:1, L0753:1,L0755:1, L07591 and LOS92:1. 8 HAJBV67 866415 18 AR039:11, AR219:10,AR316:9, AR218:9, AR089:9, AR270:8, AR282:8, AR283:8, AR269:8, AR248:7,AR299:7, AR183:7, AR309:7, AR277:7, AR096:7, AR104:7, AR313:7, AR281:7,AR265:6, AR249:6, AR253:6, AR315:6, AR300:6, AR290:6, AR292:6, AR202:6,AR312:6, AR280:6, AR175:6, AR231:5, AR182:5, AR185:5, AR268:5, AR060:5,AR294:5, AR194:5, AR238:5, AR247:4, AR243:4, AR053:4, AR267:4, AR295:4,AR291:4, AR055:4, AR285:4, AR213:4, AR052:4, AR184:4, AR293:4, AR296:3,AR240:3, AR033:3, AR310:3, AR314:3, AR275:3, AR246:3, AR205:3, AR286:3,AR271:3, AR234:3, AR192:3, AR298:3, AR263:3, AR232:3, AR256:3, AR251:3,AR259:2, AR284:2, AR229:2, AR289:2, AR226:2, AR274:2, AR237:2, AR258:2,AR179:2, AR177:2, AR233:2, AR273:2, AR227:2, AR204:2, AR186:1, AR061:1,AR241:1 L0754:9, S0444:6, S0442:5, S0358:5, H0622:5, H0624:4, H0040:4,L0659:4, H0144:4, H0521:4, H0171:3, L3499:3, L2630:3, H0046:3, H0658:3,H0555:3, H0436:3, L0758:3, S0434:3, H0543:3, S0418:2, S0360:2, S0222:2,L2499:2, H0013:2, H0156:2, H0575:2, H0615:2, H0674:2, H0616:2, H0551:2,H0412:2, H0623:2, S0440:2, H0647:2, S0422:2, H0529:2, L0666:2, L2263:2,S0374:2, S0380:2, S0146:2, L0740:2, L0731:2, L0759:2, S0436:2, L0362:2,H0556:1, L3644:1, S0114:1, T0049:1, L0002:1, L2910:1, S0282:1, L2300:1,S0356:1, S0354:1, S0408:1, S0410:1, L3709:1, H0637:1, H0742:1, H0722:1,S0046:1, S0132:1, S0300:1, L2744:1, L0717:1, H0411:1, H0431:1, H0586:1,H0587:1, L2491:1, L2647:1, H0036:1, H0004:1, S0010:1, H0318:1, H0581:1,H0251:1, H0596:1, L0471:1, H0024:1, H0014:1, H0375:1, H0266:1, S0003:1,S0214:1, H0688:1, T0023:1, H0553:1, L0055:1, H0032:1, S0036:1, H0090:1,H0591:1, H0038:1, T0067:1, H0477:1, H0488:1, H0059:1, H0561:1, L0369:1,L3905:1, L0662:1, L0766:1, L0388:1, L0774:1, L0775:1, L0606:1, L0661;1,L0526;1, L0809:1, L0665:l, L2653:1, L3665:1, L3811:1, H0519:1, S0126:1,H0683:1, H0684:1, H0659:1, H0672:1, H0710:1, H0522:1, S3014:1, S0028:1,L0752:1, H0595:1, S0394:1, L0596:1, L0589:1, L0485:1, L0595:1, H0667:1,S0242:1, S0194:1, H0423:1, H0422d, S0384:1, H0506:1 and H0352:1. 9HAJCH70 827275 19 H0561:1 10 HAOAG15 852204 20 AR169:4, AR241:3,AR172:3, AR206:3, AR263:3, AR207:3, AR176:3, AR235:3, AR168:2, AR183:2,AR297:2, AR166:2, AR163:2, AR282:2, AR171:2, AR193:2, AR178:2, AR181:2,AR162:2, AR274:2, AR182:2, AR298:2, AR217:2, AR224:2, AR312:2, AR053:2,AR287:2, AR254:2, AR295:2, AR239:2, AR205:1, AR293:1, AR216:1, ARL7S:1,AR238:1, AR285:1, AR316:1, AR277:1, AR033:1, AR179:1, AR267:1, AR291:1,AR288:1, AR289:1, AR089:1 L0759:3, S0314:2, L0744:2, L0756:2, L0755:2,S0046:1, H0391:1, H0052:1, H0050:1, S0318:1, S0338:1, S0312:1, L0766:1and H0144:1. 11 HATCD80 826098 21 AR316:50, AR055:4, AR277:3, AR060:3,AR300:3, AR282:3, AR283:2, AR218:2, AR039:1, AR104:1, AR089:1, AR240:1,AR299:1, AR185:1 H0156:1 and H0038:1. 12 HATCI03 580805 22 AR313:42,AR039:30, AR299:20, AR096:19, AR185:19, AR277:18, AR300:18, AR089:17,AR219:15, AR240:14, AR218:13, AR316:12, AR104:10, AR060:10, AR282:8,AR055:7, AR283:5 S6026:1, H0156:1 and S0426:1. 13 HBAGD86 838799 23AR219:7, AR218:4, AR313:4, AR104:4, AR039:3, AR299:3, AR282:2, AR300:2,AR096:2, AR316:2, AR277:1, AR240:1, AR089:1 L0809:4, L0766:3, L0439:3,H0624:2, H0411:2, L0794:2, L0749:2, L0756:2, L0005:1, L3649:1, S0476:1,H0599:1, L0471:1, S0051:1, T0010:1, H0266:1, S0150:1, S0422:1, L0637:1,L0765:1, L0803:1, L0783:1, L5622:1, H0144:1, H0672:1, S0392:1, L0748:1,L0754:1, L0779:1, L0777:1, L0731:1 and L0759:1. 14 HBHAA05 603174 24AR313:74, AR039:45, AR299:34, AR089:33, AR185:31, AR277:29, AR096:26,AR300:26, AR240:22, AR316:20, AR218:17, AR060:15, AR219:14, AR104:14,AR282:11, AR055:9, AR283:6 S0029:1 15 HBHAA81 846465 25 AR289:34,AR291:33, AR283:32, AR055:32, AR294:26, AR266:26, AR286:26, AR256:23,AR285:21, AR293:19, AR.259:17, AR295:16, AR292:15, AR298:14, AR258:14,AR296:12, AR284:11, AR104:10, AR033:9, AR186:9, AR202:7, AR206:7,AR246:7, AR204:7, AR241:6, AR194:5, AR198:4, AR244:4, AR251:4, AR060:4,AR061:4, AR282:4, AR052:4, AR053:4, AR205:4, AR309:4, AR316:3, AR182:3,AR312:3, AR192:3, AR273:3, AR229:3, AR183:3, AR310:3, AR271:3, AR213:3,AR248:3, AR270:3, AR277:2, AR185:2, AR275:2, AR299:2, AR269:2, AR300:2,AR247:2, AR267:2, AR175:2, AR089:2, AR313:2, AR265:2, AR268:2, AR237:2,AR096:1, AR232:1, AR039:1, AR240:1, AR179:1, AR231:1, AR234:1 H0599:8,S0366:7, L0485:6, H0733:5, H0734:5, L0769:5, H0735:4, H0729:3, H0728:3,H0619:2, H0706:2, L0661:2, L0756:2, L0759:2, S0282:1, S0029:1, S0222:1,L0622:1, H0122:1, S0010:1, H0196:1, H0012:1, H0200:1, H0373:1, S6028:1,S0364:1, S0036:1, S0294:1, L0770:1, L0638:1, L5565:1, L0657:1, L0809:1,L0789:1, L0791:1, L0438:1, L0439:1, L0750:1, L0777:1, S0260:1, L0604:1and S0460:1. 16 HBIAA59 806303 26 AR313:13, AR089:13, AR299:12,AR240:11, AR104:11, AR185:11, AR039:11, AR055:10, AR096:10, AR218:9,AR060:9, AR219:7, AR316:7, AR300:7, AR282:6, AR283:5, AR277:5 L0747:13,L0757:12, L0754:8, L0749:6, L0740:5, L0731:4, H0009:3, H0051:3, L0750:3,L0756:3, L0777:3, L0752:3, S0376:2, S0360:2, H0619:2, L3388:2, H0485:2,L3653:2, S0010:2, H0052:2, H0251:2, S0022:2, H0090:2, H0494:2, L0662:2,L0794:2, L0806:2, L0776:2, L0665:2, H0144:2, S0390:2, L0748:2, L0581:2,H0265:1, H0556:1, H0716:1, S0402:1, L0808:1, S0212:1, S0001:1, H0661:1,S0358:1, S0444:1, S0046:1, S6026:1, L0717:1, H0549:1, S0222;1, H0438:1,H0592:1, H0333:1, H0632:1, H0486:1, H0013:1, H0042:1, S0049:1, H0744:1,H0545:1, H0123:1, H0081:1, H0050:1, L0471:1, H0105:1, H0012:1, H0620:1,S00S1:1, S6028:1, H0688:1, H0553:1, L0455:1, H0598:1, H0040:1, H0412:1,L0763:1, L0769:1, L0638:1, L0372:1, L0764:1, L0771:1, L0766:1, L0561:1,L0498:1, L0774:1, L0775:1, L0375:1, L0378:1, L0805:1, L0657:1, L0659:1,L0809:1, L5623:1, L0789:1, L0663:1, L0438:1, H0520:1, H0518:1, H0696:1,S3112:1, S3014:1, S0028:1, L0744:1, L0751:1, L0780:1, L0755:1, L0758:1,L0759:1, S0031:1, S0260:1, H0506:1 and H0008:1. 17 HBJAC40 841235 27AR104:23, AR060:6, AR055:6, AR283:5, AR185:5, AR282:4, AR313:4, AR299:4,AR316:4, AR277:3, AR096:3, AR240:3, AR219:2, AR089:2, AR300:2, AR039:2,AR218:2 L0439:18, H0052:12, L0741:8, L0438:6, S0051:5, H0556:4, L0769:4,L0774:4, S0474:3, H0622:3, S0036:3, L3905:3, H0261:2, H0318:2, H0194:2,L0471:2, H0538:2, L0749:2, L0757:2, L0758:2, S0436:2, L0593:2, H0624:1,H0265:1, S0342:1, H0717:1, H0650:1, H0657:1, S0212:1, S0282:1, H0730:1,S0045:1, S0476:1, H0619:1, S0222:1, H0455:1, H0559:1, H0075:1, H0253:1,H0251:1, H0544:1, L0158:1, H0012:1, S0050:1, L0163:1, H0083:1, H0594:1,H0615:1, T0006:1, H0708:1, H0087:1, H0056:1, S0038:1, H0494:1, S0450:1,S0144:1, L0770:1, L4747:1, L0639:1, L0761:1, L0775:1, L0805:1, L0635:1,L5622:1, L0788:1, S0428:1, S0044:1, L0612:1, L0742:1, L0748:1, L0779:1,L0777:1, S0011:1 and H0136:1. 18 HBJCR46 815649 28 L0794:11, L0803:10,L0779:10, H0038:9, L0777:9, L0758:9, S0358:6, L0809:5, S0408:4, H0616:4,L0748:4, L0439:4, L0591:4, S0282:3, L0789:3, L0666:3, L0438:3, L0756:3,H0036:2, H0196:2, H0046:2, H0154:2, L0163:2, H0213:2, S0036:2, L0804:2,L0774:2, L0655:2, L0656:2, S0374:2, S0126:2, S0328:2, S0152:2, H0521:2,S0406:2, L0731:2, L0588:2, L0485:2, S0026:2, H0543:2, H0170:1, H0171:1,H0713:1, S6024:1, H0650:1, H0656:1, S0116:1, H0341:1, H0638:1, S0442:1,S0354:1, S0360:1, H0580:1, S0045:1, H0619:1, H0437:1, S0222:1, H0333:1,H0574:1, H0486:1, H0575:1, H0590:1, H0618:1, H0253:1, H0318:1, H0581:1,H0230:1, H0597:1, H0544:1, H0178:1, H0123:1, H0050:1, S0050:1, H0014:1,S6028:1, H0266:1, S0003:1, H0033:1, H0032:1, H0673:1, H0598:1, H0163:1,H0040:1, H0551:1, H0623:1, H0100:1, T0041:1, H0561:1, S0438:1, H0641:1,S0344:1, S0002:1, S0426:1, L0769:1, L0800:1, L0641:1, L0766:1, L0775:1,L0375:1, L0653:1, L0634:1, L0659:1, L0783:1, L0787:1, L0663:1, L0664:1,L0665:1, H0725:1, H0670:1, H0522:1, H0436:1, H0540:1, S0027:1, L0740:1,L0751:1, L0747:1, L0749:1, L0786:1, L0780:1, L0752:1, L0757:1, L0608:1,L0604:1, S0192:1 and S0276:1. 19 HBJDW56 520401 29 AR055:8, AR060:7,AR282:6, AR104:5, AR313:5, AR185:5, AR300:4, AR089:4, AR299:4, AR240:4,AR039:4, AR219:4, AR316:3, AR096:3, AR283:3, AR218:3, AR277:2 H0318:1 20HBJEL16 847030 30 H0046:2, H0009:2, H0090:2, H0494:2, L0438:2, H0547:2,H0521:2, L0439:2, L0777:2, H0543:2, H0556:1, S0342:1, S0045:1, H0619:1,H0632:1, H0013:1, H0156:1, L0021:1, H0575:1, H0318:1, S0003:1, L0483:1,H0628:1, H0623:1, H0561:1, L0761:1 L0803:1, L0804:1, L0659:1, L0382:1,H0144:1, H0539:1, S0152:1, H0478:1, H0631:1, L0741:1, L0740:1 andL0591:1. 21 HBJIG20 866159 31 AR060:1246, AR282:1116, AR300:1098,AR055:1006, AR104:977, AR299:929, AR185:901, AR240:901, AR283:813,AR316:761, AR277:738, AR089:695, AR039:604, AR096:571, AR313:390,AR218:357, AR219:319 H0594:11, H0596:8, S0282:5, S0260:5, S0194:5,H0543:5, S0278:2, H0600:2, H0592:2, H0598:2, S0344:2, H0595:2, S0356:1,H0438:1, H0574:1, H0599:1, S0346:1, H0318:1, H0597:1, S0388:1, H0316:1,S0390:1 and H0542:1. 22 HBJKD16 853358 32 AR172:63, AR171:62, AR215:61,AR274:50, AR216:48, AR213:43, AR214:41, AR272:41, AR169:41, AR224:37,AR225:37, AR217:37, AR254:36, AR205:36, AR170:35, AR243:35, AR168:35,AR247:34, AR245:32, AR312:32, AR221:32, AR212:31, AR161:29, AR222:28,AR162:28, AR311:27, AR308:27, AR163:26, AR275:26, AR165:25, AR164:24,AR313:23, AR053:23, AR166:23, AR223:21, AR039:20, AR089:20, AR309:19,AR096:19, AR242:18, AR253:18, AR240:17, AR289:16, AR266:16, AR283:16,AR263:16, AR193:16, AR316:16, AR264:16, AR204:16, AR250:15, AR282:15,AR201:15, AR277:15, AR207:14, AR291:14, AR246:14, AR200:13, AR198:13,AR271:12, AR299:12, AR300:12, AR195:12, AR155:12, AR104:12, AR290:11,AR192:11, AR173:11, AR255:11, AR257:11, AR060:11, AR197:11, AR252:10,AR150:1O, AR297:1O, AR179:10, AR210:10, AR061:10, AR181:9, AR296:9,AR199:9, AR270:9, AR269:9, AR175:9, AR183:9, AR268:8, AR055:8, AR177:8,AR262:8, AR236:8, AR288:8, AR211:8, AR188:7, AR267:7, AR293:7, AR219:7,AR285:7, AR256:7, AR294:7, AR174:7, AR176:7, AR189:7, AR033:7, AR261:7,AR218:7, AR287:6, AR175:6, AR196:6, AR231:6, AR203:6, AR286:5, AR235:5,AR190:5, AR230:5, AR234:5, AR191:5, AR182:5, AR260:5, AR258:4, AR295:4,AR237:4, AR233:4, AR229:4, AR238:4, AR239:3, AR226:3, AR232:2, AR227:2,AR228:2 L0766:9, L0439:9, L0747:6, L2528:5, L0777:5, H0673:4, L0438:4,L0758:4, L0362:4, S0116:3, L0748:3, L0752:3, H0445:3, H0156:2, T0010:2,H0615;2, H0038:2, H0616:2, H0264:2, H0646:2, L0761:2, L0776:2, L0750:2,L0779:2, S0436:2, L0593:2, S0242:2, H0222:1, H0740:1, H0657:1, H0661:1,H0663:1, L2293:1, H0589:1, S0444:1, H0340:1, L3646:1, H0580:1, H0749:1,H0393:1, H0549:1, S0222:1, H0574:1, H0486:1, H0013:1, H0069:1, L0021:1,S0010:1, H0318:1, S0474:1, H0046:1, L0471:1, H0090:1, L0638:1, L0646:1,l0764:1, L0521:1, L0364:1, L0774:1, L0659:1, L0543:1, L5622:1, L0792:1,L0666:1, L0664:1, L0665:1, S0428:1, L2657:1, L2652:1, L3663:1, L2262:1,H0435:1, L3832:1, L0741:1, L0749:1, S0434:1, L0588:1, H0422:1, L0698:1and L2359:1. 23 HBMTY48 637521 33 AR241:34, AR313:20, AR039:19,AR192:19, AR182:18, AR198:16, AR275:14, AR271:13, AR312:13, AR184:13,AR186:12, AR274:12, AR290:11, AR268:11, AR185:11, AR089:10, AR204:10,AR267:10, AR096:10, AR270:9, AR299:9, AR052:9, AR240:9, AR213:9,AR273:9, AR243:9, AR291:8, AR247:8, AR300:8, AR269:8, AR053:8, AR293:8,AR194:8, AR258:8, AR206:8, AR316:8, AR277:7, AR104:7, AR060:7, AR292:6,AR218:6, AR246:6, AR309:6, AR183:6, AR263:6, AR244:6, AR202:6, AR205:5,AR233:5, AR296:5, AR282:5, AR177:5, AR294:5, AR289:5, AR033:5, AR061:5,AR285:5, AR219:5, AR175:5, AR310:4, AR295:4, AR055:4, AR298:4, AR259:4,AR249:4, AR266:4, AR229:4, AR179:3, AR265:3, AR256:3, AR248:2, AR283:2,AR284:2, AR280:2, AR286:2, AR226:1, AR237:1 S0116:1, H0591:1, L2270:1,L0766:1, L2657:1 and H0690:1. 24 HBMUH74 866160 34 AR218:12, AR055:8,AR060:7, AR104:7, AR219:5, AR240:5, AR299:5, AR096:4, AR316:4, AR300:4,AR039:4, AR089:3, AR283:3, AR185:3, AR313:3, AR282:2, AR277:2 L0754:3,L0777:3, L0439:2, S0116:1, H0341:1, H0661:1, H00381, H0412:1, L0761:1,L0667:1, L0764:1, L0788:1, H0435:1, L0749:1, L0779:1 and LO758:1. 25HBQAB79 810542 35 AR055:7, AR218:7, AR060:6, AR039:6, AR300:5, AR185:5,AR313:5, AR240:4, AR299:4, AR089:4, AR096:3, AR316:3, AR283:3, AR104:2,AR219:2, AR277:2, AR282:1 H0229:1 26 HBXCX15 637542 36 S0038:3, H0438:1,L0363:1 and S0053:1. 27 HCDCY76 837972 37 AR219:7, AR218:6, AR055:2,AR282:2, AR060:2, AR299:1, AR104:1, AR185:1, AR240:1, AR277:1 L1430:5,L0770:2, L0754:2, L0747:2, L0777:2, S0360:1, S0045:1, H0486:1, H0616:1,L0803:1, L0775:1, L0783:1, L0787:1, L0789:1, L0750:1, S0194:1 andS0276:1. 28 HCEDR26 771144 38 AR313:59, AR039:47, AR277:33, AR299:28,AR185:25, AR096:23, AR089:23, AR300:20, AR219:19, AR240:17, AR316:16,AR104:15, AR282:13, AR218:13, AR060:13, AR283:10, AR055:10 H0052:2,H0018:1, H0264:1 and L0700:1. 29 HCEEU18 688041 39 AR313:46, AR039:35,AR299:24, AR219:21, AR277:21, AR089:20, AR096:19, AR185:19, AR218:16,AR316:14, AR300:13, AR104:13, AR240:12, AR060:11, AR282:10, AR055:9,AR283:5 H0052:1 30 HCEGX05 827060 40 AR219:16, AR104:13, AR218:13,AR089:11, AR185:9, AR313:9, AR299:8, AR240:8, AR096:8, AR316:8, AR055:7,AR039:6, AR060:6, AR300:6, AR283:5, AR277:4, AR282:4 L0766:11, L0748:5,L0757:4, L0662:3, H0587:2, L3816:2, L0041:2, H0039:2, L0659:2, L0438:2,H0672:2, H0521:2, L0750:2, L0758:2, L0596:2, L0589:2, L0605:2, H0265:1,H0341:1, H0728:1, S0222:1, H0600:1, L0623:1, H0069:1, H0052:1, H0569:1,S0388:1, T0010:1, L0055:1, L0456:1, H0560:1, H0641:1, S0426:1, L0770:1,L0769:1, L5575:1, L0794:1, L0776:1, L0783:1, L0382:1, L0666:1, L0663:1,S0052:1, S0216:1, H0702:1, L3825:1, L3828:1, H0670:1, H0539:1, H0522:1,S0406:1, S0390:1, L0743:1, L0744:1, L0439:1, L0740:1, L0747:1, L0779:1,L0777:1, H0445:1, S0436:1, H0542:1, H0423:1 and H0422:1. 31 HCFLN88610000 41 S0410:22, L0770:9, L0748:9, L0769:7, L0776:6, L0659:6,H0424:5, L0761:5, L0731:5, H0486:4, L0803:4, L0809:4, L0666:4, H0696:4,L0754:4, L0779:4, L0758:4, H0729:3, H0618:3, H0135:3, L0637:3, L0771:3,L0766:3, L0805:3, L0665:3, L0751:3, H0542:3, H0341:2, H0402:2, S0358:2,S0376:2, S0360:2, H0747:2, S0132:2, L3109:2, L0717:2, H0592:2, H0253:2,S0010:2, H0052:2, H0545:2, H0050:2, H0617:2, H0087:2, H0551:2, H0100:2,H0560:2, L0763:2, L5565:2, L0646:2, L0764:2, L0655:2, L0663:2, L2260:2,S0374:2, H0414:2, S0406:2, H0436:2, L0743:2, L0740:2, L0749:2, L0755:2,L0757:2, L0759:2, H0445:2, H0136:2, H0543:2, H0423:2, H0352:2, H0170:1,H0171:1, H0225:1, H0713:1, S0218:1, L0785:1, H0692:1, S0212:1, H0483:1,H0254:1, H0305:1, S0356:1, S0442:1, S0444:1, S0408:1, H0619:1, H0393:1,H0406:1, H0370:1, H0249:1, H0101:1, H0250:1, S0280:1, H0599:1, H0575:1,H0706:1, T0048:1, H0318:1, S0474:1, H0581:1, T0115:1, H0009:1, H0572:1,H0024:1, S0051:1, H0271:1, H0288:1, T0006:1, H0213:1, H0553:1, H0644:1,S0364:1, H0163:1, H0090:1, H0264:1, H0488:1, S0112:1, H0494:1, H0652:1,S0344:1, S0002:1, S0426:1, L4497:1, L5575:1, L3905:1, L5566:1, L0772:1,L0641:1, L0645:1, L0773:1, L0650:1, L0774:1, L0775:1, L0378:1, L0806:1,L0783:1, L5622:1, L0790:1, L0664:1, L3827:1, H0547:1, H0519:1, S0126:1,H0711:1, H0672:1, S0330:1, H0521:1, S0392:1, S0037:1, L0742:1, L0439:1,L0745:1, L0747:1, L0750:1, L0777:1, S0436:1, L0485:1, L0608:1, S0011:1,H0653:1 and H0422:1. 32 HCHAB84 834326 42 AR313:37, AR039:34, AR104:24,AR300:24, AR277:23, AR096:23, AR185:20, AR089:20, AR299:19, AR219:18,AR218:17, AR316:16, AR240:16, AR282:13, AR283:9, AR060:8, AR055:7S0354:9, S0358:3, H0494:3, S04762, S0474:2, 50438:2, H0519:2, H0521:2,L0754:2, H0170:1, S0040:1, S0114:1, H0484:1, H0483:1, H0255:1, S0376:1,S0444:1, S0408:1, S0046:1, H0619:1, H0549:1, H0042:1, H058:1, H0052:1,H0083:1, S0440:1, L0773:1, L0517:1, L0383:1, S0374:1, S0152:1, S3014:1,L0751:1, L0759:1, S0434:1, S0436:1, H0543:1 and H0422:1. 33 HCMSX51788643 43 L0740:16, L0745:7, L0439:6, L0438:4, H0547:4, L0750:4,L0759:4, H0619:3, H0618:3, L0770:3, L3828:3, L0749:3, L0758:3, H0393:2,H0599:2, H0083:2, H0124:2, H0623:2, H0100:2, L3905:2, L0794:2, L0809:2,L3825:2, L3829:2, S0406:2, S0027:2, L0743:2, L0746:2, L0777:2, L0603:2,S0040:1, L2879:1, L2906:1, S0420:1, S0442:1, S0358:1, L3311:1, L3485:1,H0261:1, H0392:1, H0013:1, H0250:1, H0590:1, H0196:1, H0545:1, H0046:1,H0123:1, H0620:1, S0051:1, S0250:1, H0617:1, S0036:1, H0135:1, H0634:1,H0087:1, H0269:1, H0561:1, H0509:1, H0646:1, S0426:1, L0763:1, L0769:1,L3904:1, L0662:1, L0363:1, L0767:1, L0768:1, L0650:1, L0375:1, L0806:1,L0776:1, L0657:1, L0787:1, L4559:1, L0664:1, L2260:1, H0520:1, L3831:1,H0670:1, H0518:1, L3834:1, H0704:1, H0436:1, L0747:1, L0780:1, L0608:1,L0595:1 and H0423:1. 34 HCNCO11 775086 44 AR055:2, AR060:2, AR277:1,AR282:1 H0597:1 35 HCNSD29 862314 45 AR252:128, AR253:67, AR245:63,AR272:55, AR308:49, AR246:47, AR263:46, AR212:40, AR053:37, AR243:35,AR312:34, AR254:33, AR275:33, AR205:33, AR309:32, AR264:31, AR250:31,AR197:31, AR271:29, AR224:26, AR195:26, AR311:26, AR200:26, AR223:26,AR201:25, AR198:25, AR219:23, AR274:22, AR210:22, AR172:21, AR218:21,AR222:21, AR225:20, AR221:20, AR268:19, AR1O4:19, AR096:18, AR240:17,AR188:17, AR313:17, AR199:17, AR213:17, AR203:16, AR242:16, AR039:15,AR316:15, AR170:15, AR193:15, AR180: 15, AR165:14, AR269:14, AR192:14,AR204:14, AR164:14, AR166: 14, AR211:13, AR033:13, AR168:13, AR270:13,AR207:12, AR175:12, AR290:12, AR169:12, AR089:12, AR183:12, AR247:12AR176.11, AR171:11, AR267:11, AR178:11, AR162:11, AR161:11, AR217:11,AR163:10, AR174:10, AR229:10, AR291:10, AR173:10, AR189:9, AR214:9,AR266:9, AR255:9, AR181:8, AR196:8, AR177:8, AR297:8, AR289:8, AR299:8,AR179:8, AR190:8, AR191:8, AR300:8, AR060:8, AR182:8, AR238:7, AR216:7,AR215:7, AR257:7, AR261:7, AR262:7, AR282:7, AR296:7, AR185:7, AR293:7,AR235:6, AR295:6, AR283:6, AR231:6, AR055:6, AR288:6, AR285:6, AR287:6,AR234:6, AR258:6, AR237:5, AR239:5, AR236:5, AR256:5, AR277:5 , AR286:5,AR228:5, AR230:4, AR294:4, AR226:4, AR233:4, AR260:4, AR232:4, AR061:3,AR227:3 L0648:2, L0768:2, L0766:2, L0748:2, L0588:2, H0125:1, S0468:1,H0497:1, H0486:1, H0744:1, H0231:1, H0266:1, H0202:1, H0641:1, S0422:1,L0638:1, L0644:1, L5572:1, L0662:1, L0650:1, L0807:1, L0657:1, L0663:1,L0665:1, H0519:1, L0759:1, S0026:1 and L0718:1. 36 HCQBH72 637548 46AR055:2, AR060:2, AR299:2, AR089:2, AR219:1, AR185:1, AR039:1, AR283:1,AR104:1 L0520:4, L0754:2, H0263:1, H0272:1 and H0555:1. 37 HCQCC96845066 47 AR252:46, AR197:44, AR204:38, AR195:35, AR253:34, AR178:31,AR230:31, AR254:31, AR233:29, AR250:28, AR18O028, AR198:28, AR266:26,AR243:26, AR193:24, AR239:23, AR061:23, AR267:23, AR201:23, AR227:22,AR229:22, AR228:22, AR237:21, AR162:21, AR181:21, AR163:21, AR170:21,AR161:20, AR257:20, AR192:20, AR226:20, AR176:19, AR234:19, AR171:18,AR183:18, AR245:18, AR271:18, AR182:17, AR258:17, AR270:17, AR179:17,AR275:17, AR238:16, AR231:16, AR296:16, AR261:16, AR033:16, AR174:15,AR185:15, AR255:15, AR164:15, AR262:15, AR207:15, AR053:15, AR165:15,AR272:14, AR256:14, AR039:14, AR269:14, AR242:14, AR166:14, AR205:14,AR175:14, AR246:14, AR300:13, AR203:13, AR289:12, AR236:12, AR104:12,AR316:11, AR232:11, AR293:11, AR055:11, AR287:11, AR169:11, AR260:11,AR235:11, AR168:11, AR089:11, AR173:10, AR308:10, AR286:10, AR268:10,AR291:10, AR060:10, AR297:10, AR313:10, AR288:10, AR212:10, AR213:10,AR299:10, AR177:10, A2R188:9, AR096:9, AR190:9, AR191:9, AR294:9,AR282:9, AR285:9, AR.283:9, AR172:8, AR277:8, AR189:8, AR247:8, AR309:8,AR312:8, AR274:8, AR240:7, AR218:7, AR264:7, AR210:6, AR200:6, AR295:6,AR290:6, AR219:6, AR215:6, AR199:5, AR263:5, AR196:5, AR223:5, AR311:5,AR216:5, AR214:4, AR224:4, AR225:4, AR211:4, AR217:4, AR221:2, AR222:2S0360:5, L0748:5, L0766:3, H0657:2, L3388:2, H0581:2, H0596:2, H0563:2,S0003:2, H0328:2, H0670:2, L0756:2, S0436:2, S0026:2, H0170:1, H0556:1,H0344:1, H0650:1, H0656:1, H0638:1, S0420:1, H0675:1, S0007:1, H0574:1,H0632:1, H0013:1, H0036:1, S0010:1, H0318:1, H0052:1, H0251:1, H0150:1,H0050:1, H0090:1, H0038:1, S0440:1, H0130:1, S0142:1, S0422:1, H0529:1,L0803:1, L0659:1, L5623:1, L0666:1, S0428:1, S0126:1, H0689:1, H0648:1,H0672:I, S0330:1, H0539:1, S0378:1, H0521:1, H0522:1, H0478:1, L0744:1,L0754:1, L07791, L0752:1, S0260:1, H0445:1, H0343:1, H0595:1, S0434:1,H0423:1 and S0424:1. 38 HCQCJ56 832157 48 AR055:8, AR060:5, AR104:4,AR240:3, AR218:3, AR299:3, AR300:3, AR185:3, AR277:3, AR089:2, AR283:2,AR282:2, AR039:2, AR316:2, AR096:2, AR219:2 L0779:4, L0777:3, H0050:2,H0670:2, L0748:2, L0717:1, H0596:1, L0641:1, L0794:1, L0803:1, L0774:1,L0809:1 and L0749:1. 39 HCUDD64 835082 49 AR282:3, AR219:3 H0052:3,S3012:2, L0754:2, H0402:1, H0413:1, S0374:1, L0438:1, L0748:1 andL0740:1. 40 HCWAE64 535893 50 AR277:7, AR282:1 H0305:1 41 HDPDJ58 58726551 AR263:8, AR249:6, AR053:5, AR270:5, AR312:5, AR039:4, AR309:4,AR096:4, AR052:4, AR198:4, AR183:4, AR253:4, AR313:4, AR282:4, AR243:3,AR269:3, AR184:3, AR192:3, AR267:3, AR268:3, AR213:3, AR316:3, AR290:3,AR310:2, AR240:2, AR275:2, AR273:2, AR186:2, AR238:2, AR298:2, AR206:2,AR234:2, AR277:2, AR177:2, AR292:2, AR226:1, AR060:1, AR237:1, AR296:1,AR205:1, AR299:1, AR033:1, AR294:1, AR293:1, AR291:1, AR231;1, AR175:1,AR182:1, AR185:1, AR284:1, AR218:1 L0766:14, H0457:10, H0486:4, H0581:4,S0406:4, H0422:4, H0171:3, L0655:3, H0521:3, L0779:3, H0749:2, H0156:2,H0090:2, H0551:2, L0598:2, L0666:2, L0438:2, L0748:2, L0756:2, L0777:2,T0002:1, H0656:1, S0212:1, H0662:1, H0638:1, S0442:1, S0140:1, H0747:1,H0261:1, H0587:1, L3816:1, H0574:1, L0586:1, L0022:1, H0318:1, H0123:1,L0471:1, H0039:1, H0591:1, T0041:1, S0344:1, S0426:1, UNKWN:1, L0794:1,L0387:1, L0776:1, L0606:1, L0659:1, L0367:1, L0792:1, L0793:1, H0690:1,H0539:1, H0436:1, L0439:1, L0780:1, L0755:1, L0759:1, H0445:1, H0423:1and H0506:1. 42 HDPFU43 790189 52 AR277:53, AR283:13, AR096:12,AR240:12, AR316:11, AR219:11, AR218:10, AR104:9, AR282:9, AR299:8,AR039:8, AR313:8, AR185:8, AR300:7, AR060:7, AR055:7, AR089:7 H0585:8,L3388:8, S0474:7, H0622:4, H0141:3, H0553:3, S0126:3, H0539:3, L0750:3,H0556:2, H0717:2, H0581:2, S0440:2, S0344:2, L0771:2, L0774:2, L0664:2,S0380:2, H0521:2, L0751:2, L0755:2, L3643:1, H0650:1, H0306:1, S0420:1,L0617:1, S0444:1, S0360:1, H0580:1, S0046:1, H0619:1, H0549:1, H0486:1,T0039:1, L0021:1, H0274:1, H0457:1, H0012:1, H0620:1, S0003:1, S0214:1,H0615:1, H0628:1, H0087:1, H0551:1, S0438:1, S0422:1, H0529:1, L0770:1,L0761:1, L0767:1, L0768:1, L0804:1, L0515:1, L0809:1, H0703:1, H0711:1,H0672:1, S0378:1, H0522:1, H0696:1, H0555:1, S3014:1, L0754:1, L0747:1,L0749:1, L0731:1, H0445:1, S0436:1, L0581:1, S0026:1, H0543:1 andH0423:1. 43 HDPGE24 801947 53 H0555:8, S0002:7, L0748:6, H0556:5,H0179:5, L0369:5, S0222:4, S0474:4, S0045:3, H0427:3, H0599:3, H0575:3,H0271:3, H0628:3, H0598:3, S0426:3, L0766:3, L0581:3, H0265:2, S0114:2,S0212:2, H0402:2, S0442:2, S0354:2, S0132:2, H043 1:2, H0370:2, H0632:2,H0581:2, H0196:2, H0050:2, H0124:2, L0665:2, H0521:2, S0390:2, S0028:2,L0777:2, H0444:2, S0436:2, H0423:2, L3643:1, S0040:1, L0002:1, H0381:1,S0116:1, H0255:1, H0662:1, S0360:1, H0676:1, H0580:1, H0729:1, H0722:1,H0728:1, S0046:1, H0749:1, S0300:1, L0717:1, L3388:1, H0586:1, H0333:1,H0486:1, H0706:1, H0036:1, T0048:1, H0318:1, H0251:1, H0309:1, H0121:1,H0544:1, S0050:1, H0375:1, H0266:1, S0003:1, S0214:1, H0252:1, H0031:1,H0644:1, H0708:1, H0400:1, H0063:1, H0264:1, S0038:1, H0280:1, H0334:1,H0625:1, S0440:1, H0509:1, H0132:1, 50210:1, L0803:1, L0525:1, L0555:1,L0529:1, L0367:1, L0532:1, S0052:1, S0428:1, S0216:1, H0547:1, H0519:1,S0126:1, H0134:1, S0406:1, H0727:1, H0345:1, S0037:1, L0740:1, L0749:1,S0031:1, H0445:1, H0707:1, L0605:1, L0604:1, L0601:1 and H0543:1. 44HDPIU94 813352 54 AR055:17, AR277:13, AR060:12, AR316:9, AR219:8,AR240:8, AR089:8, AR300:8, AR218:8, AR039:7, AR283:7, AR096:6, AR282:5,AR104:5, AR185:4, AR299:4, AR313:2 L0748:6, L0666:5, L0665:5, L0768:4,L0777:4, L0595:4, H0352:4, S0045:3, H0124:3, L0774:3, S0028:3, L0439:3,L0756:3, L0592:3, S0376:2, S0360:2, H0619:2, S0222:2, L3816:2, H0635:2,H0036:2, H0052:2, H0046:2, L0041:2, S0312:2, H0551:2, L3815:2, L0764:2,L0663:2, H0144:2, L3825:2, L0751:2, L0754:2, L0745:2, L0731:2, L0589:2,H0653:2, H0136:2, H0216:2, H0624:1, S6024:1, S0430:1, H0656:1, H0255:1,S0046:1, H0747:1, H0645:1, L2759:1, H0013:1, H0156:1, H0575:1, H0050:1,S0050:1, H0373:1, H0687:1, S0314:1, S0250:1, H0031:1, H0135:1, H0634:1,H0616:1, H0380:1, H0264:1, H0433:1, H0059:1, L0351:1, S0422:1, L0800:1,L0662:1, L0626:1, L0766:1, L0803:1, L0375:1, L0655:1, L0659:1, L0783:1,L0809:1, L0664:1, L2263:1, L2258:1, L2259:1, H0726:1, L3826:1, L3827:1,H0648:1, S0152:1, L3833:1, H0521:1, S0390:1, S3014:1, S0027:1, L0749:1,L0750:1, L0780:1, L0759:1, L0759:1, S0260:1 and L0366:1. 45 HDPIY31886159 55 AR214:26, AR263:25, AR224:21, AR222:20, AR264:20, AR223:19,AR169:18, AR221:18, AR217:18, AR171:17, AR172:17, AR195:17, AR207:16,AR170:16, AR311:16, AR235:16, AR215:16, AR225:16, AR168:15, AR216:15,AR165:14, AR197:14, AR164:14, AR162:14, AR308:13, AR089:13, AR161:13,AR166:13, AR212:13, AR192:13, AR193:13, AR163:13, AR213:12 AR033:12,AR309:12, AR242:12, AR053:11, AR245:11, AR312:11, AR210:10, AR282:10,AR253:10, AR261:10, AR254:10 AR277:10, AR198:10, AR104:10, AR295:10,AR288:9, AR240:9, AR316:9, AR299:9, AR219:9, AR196:9, AR205:9, AR271:9,AR252:9, AR285:9, AR250:9, AR297:9, AR272:8, AR060:8, AR096:8, AR177:8,AR269:8, AR274:8, AR246:8, AR313:8, AR236:8, AR211:8, AR185:8, AR286:8,AR039:7, AR291:7, AR287:7, AR275:7, AR200:7, AR300:7, AR055:7, AR229:7,AR181:7, AR283:7, AR189:7, AR218:7, AR175:7, AR174:7, AR247:7, AR238:7,AR199:6, AR289:6, AR188:6, AR293:6, AR243:6, AR191:6, AR173:6, AR266:6.AR262:6, AR204:6, AR270:6, AR226:6, AR258:6, AR201:6, AR176:6, AR268:5,AR296:5, AR257:5, AR183:5, AR182:5, AR234:5, AR231:5, AR180:5, AR255:5,AR230:5, AR178:5, AR256:5, AR290:5, AR190:5, AR239:5, AR232:5, AR260:4,AR227:4, AR203:4, AR294:4, AR267:4, AR061:4, AR179:4, AR237:4, AR233:3,AR228:3 L0439:56, L0438:20, H0556:7, H0052:7, L0776:5, S0222:4, H0438:4,S0418:3, S0278:3, L0770:3, L0771:3, L0743:3, L0366:3, H0265:2, S0040:2,L0415:2, S0045:2, H0619:2, H0492:2, H0486:2, H0581:2, H0620:2, H0266:2,H0604:2, H0031:2, H0100:2, L0351:2, H0144:2, L0352:2, H0672:2, H0521:2,L0756:2, L0777:2, L0731:2, H0624:1, H0140:1, H0583:1, H0255:1, H0402:1,H0305:1, H0458:1, S0420:1, S0354:1, S0358:1, H0645:1, S6022:1, H0392:1,H0643:1, H0559:1, H0013:1, H0069:1, H0156:1, H0590:1, S0346:1, H0085:1,H0544:1, H0545:1, H0439:1, H0150:1, H0041:1, S0388:1, S0051:1, T0010:1,H0271:1, H0416:1, H0188:1, H0288:1, S0022:1, T0006:1, H0213:1, H0628:1,H0617:1, H0135:1, H0087:1, H0551:1, H0477:1, H0059:1, S0038:1, L0435:1,T0042:1, H0494:1, S0344:1, S0426:1, L0640:1, L0769:1, L0638:1, L0761:1,L0642:1, L0764:1, L0768:1, L0794:1, L0803:1, L0375:1, L0806:1, L0805:1,L0655:1, L0659:1, L0809:1, L0789:1, L0665:1, H0519:1, S0126:1, H0690:1,H0682:1, S0330:1, H0539:1, H0522:1, S0037:1, L0751:1, L0754:1, L0746:1,L0749:1, L0786:1, L0779:1, H0445:1, L0596:1, H0542:1 and H0543:1. 46HDPOC24 777493 56 H0585:26, H0141:12, L0666:9, L0754:9, L0755:9,S0212:6, L0663:5, L0743:5, S0356:4, H0587:4, H0553:4, L0657:4, L0382:4,L0740:4, L0747:4, S0045:3, S0046:3, H0024:3, L0771:3, L0648:3, L0662:3,L0659:3, L0664:3, S0126:3, H0522:3, L0748:3, L0777:3, L0757:3, S0192:3,S0040:2, S0420:2, S0442:2, S0358:2, S0476:2, H0550:2, H0497:2, H0250:2,H0575:2, H0052:2, H0546:2, H0266:2, H0100:2, H0646:2, S0002:2, L0763:2,L0649:2, L0803:2, L0775:2, L0653:2, L0517:2, L0809:2, L0790:2, L0665:2,H0660:2, S0380:2, H0521:2, S3014:2, S0028:2, L0751:2, S0436:2, H0665:2,S0430:1, H0341:1, S0282:1, H0664:1, S0418:1, S0354:1, S0444:1, H0549:1,S0222:1, H0600:1, H0333:1, H0618:1, H0253:1, S0474:1, H0581:1, H0235:1,H0597:1, H0545:1, H0009:1, H0081:1, H0620:1, H0023:1, H0687:1, S0250:1,L0483:1, T0006:1, L0055:1, H0087:1, H0551:1, H0379:1, H0264:1, H0494:1,H0625:1, S0352:1, H0641:1, H0529:1, L0371:1, L0769:1, L5575:1, L3905:1,L5566:1, L0772:1, L0800:1, L0764:1, L0773:1, L0794:1, L0386:1, L0378:1,L0806:1, L0807:1, L0792:1, L0565:1, S0310:1, H0519:1, H0682:1, H0684:1,H0670:1, H0672:1, S0328:1, S0330:1, S0332:1, H0478:1, S0432:1, S3012:1,S0390:1, S0206:1, L0742:1, L0756:1, L0779:1, H0707:1, S0434:1, L0596:1,H0668:1, S0242:1, H0506:1 and H0008:1. 47 HDPPD93 637588 57 AR202:68,AR194:68, AR281:64, AR244:59, AR315:56, AR205:52, AR246:50, AR280:49,AR283:45, AR314:39, AR271:38, AR232:37, AR243:37, AR241:35, AR316:34,AR282:33, AR204:33, AR263:32, AR089:32, AR192:32, AR265:31, AR277:31,AR206:30, AR219:29, AR310:29, AR033:29, AR096:29, AR313:28, AR299:28,AR240:26, AR247:26, AR273:24, AR300:24, AR198:24, AR295:24, AR274:24,AR218:24, AR039:23, AR275:23, AR055:23, AR213:23, AR104:22, AR251:22,AR238:20, AR177:20, AR312:20, AR060:19, AR226:19, AR052:19, AR231:18,AR053:18, AR309:18, AR234:18, AR227:18, AR185:17, AR292:17, AR237:17,AR229:16, AR258:16, AR183:16, AR175:15, AR294:14, AR256:13, AR259:13,AR233:13, AR293:11, AR186:11, AR253:10, AR061:10, AR266:10, AR267:9,AR285:8, AR248:8, AR270:8, AR296:8, AR284:7, AR179:7, AR289:7, AR249:7,AR268:6, AR269:6, AR291:6, AR184:6, AR298:5, AR286:5, AR182:5, AR290:4L0794:6, L0748:6, H0556:5, L0771:5, H0052:4, L0756:4, L0596:4, H0265:3,H0341:3, H0587:3, L0662:3, L0803:3, L0790:3, S0152:3, L0750:3, S0114:2,S0360:2, H0318:2, L0471:2, L0369:2, L0763:2, L0770:2, L0764:2, L0766:2,L0774:2, L0378:2, L0789:2, L0666:2, L3825:2, H0547:2, L0747:2, L0777:2,L0581:2, H0543:2, H0422:2, S0218:1, H0255:1, S0418:1, S0354:1, S0376:1,S0408:1, L3649:1, S0045:1, H0747:1, H0619:1, L0717:1, S0222:1, H0431:1,H0586:1, H0013:1, H0069:1, S0049:1, H0009:1, H0071:1, H0083:1, H0428:1,T0006:1, H0424:1, H0213:1, H0644:1, H0628:1, H0135:1, H0163:1, H0616:1,H0413:1, H0059:1, H0561:1, S0448:1, H0647:1, L3818:1, S0002:1, L0769:1,L0500:1, L0363:1, L0767:1, L0768:1, L0649:1, L0804:1, L0806:1, L0657:1,L0512:1, L0659:1, L0384:1, L0647:1, L5622:1, L5623:1, L0664:1, L0665:1.S0374:1, L3828:1, S0126:1, H0711:1, H0658:1, H0666:1, H0539:1, H0753:1,H0521:1, H0522:1, S0406:1, H0555:1, H0436:1, L0439:1, L0749:1, S0031:1,L0595:1, H0136:1, H0542:1, H0423:1, S0424:1 and H0352:1. 48 HDPPQ30684292 58 H0542:4, S0250:3, H0521:3, H0522:3, H0485:2, H0486:1, H0494:1and H0543:1. 49 HDQHM36 852328 59 AR313:50, AR039:48, AR277:28,AR089:26, AR096:26, AR300:25, AR185:23, AR299:22, AR240:17, AR316:17,AR104:15, AR219:12, AR282:12, AR.218:12, AR060:10, AR055:6, AR283:5H0521:2 50 HDTFX18 801957 60 AR313:6, AR277:5, AR039:4, AR096:4,AR055:3, AR283:3, AR300:3, AR282:3, AR316:3, AR104:2, AR299:2, AR240:2,AR060:2, AR185:2, AR089:2, AR218:1 L0748:2, L0731:2, H0486:1, H0634:1,L0766:1, L0809:1, L0750:1 and L0777:1. 51 HE2CM39 553651 61 AR277:46,AR283:32, AR219:30, AR313:29, AR218:25, AR316:25, AR089:24, AR299:23,AR104:23, AR282:23, AR055:21, AR300:20, AR185:18, AR039:18, AR096:17,AR240:17, AR060:13 L0759:4, L0657:3, L0789:3, L0439:3, L0752:3, L0758:3,S0360:2, L0805:2, L0438:2, L0750:2, L0777:2, H0423:2, H0171:1, H0638:1,H0351:1, H0178:1, H0606:1, L0625:1, L0769:1, L0771:1, L0662:1, L0794:1,L0803:1, L0804:1, L0650:1, L0774:1, L0659:1, L0809:1, L0663:1, H0436:1,L0748:1, L0740:1, H0445:1, L0604:1 and H0422:1. 52 HE2HC60 753265 62AR277:44, AR283:36, AR219:36, AR218:34, AR055:31, AR316:29, AR313:24,AR089:24, AR104:23, AR282:22, AR299:21, AR039:19, AR240:19, AR096:18,AR185:18, AR300:18, AR060:14 L0439:13, L0777:9, L0717:8, L0748:6,L0659:5, L0747:4, H0318:3, L0665:3, L0779:3, H0170:2, H0212:2, L0455:2,S0422:2, L0764:2, L0662:2, L0768:2, L0766:2, L0775:2, L0655:2, L0809:2,H0520:2, H0672:2, L0746:2, L0755:2, L0758:2, L0759:2, L0595:2, H0624:1,H0171:1, H0685:1, H0661:1, H0402:1, S0408:1, L3646:1, S0046:1, H0333:1,T0109:1, H0013:1, S0280:1, L0021:1, H0590:1, H0581:1, H0374:1, H0596:1,L0471:1, H0014:1, S0051:1, S0003:1, H0328:1, H0617:1, H0040:1, H0412:1,H0494:1, H0641:1, L0761:1, L0645:1, L0773:1, L0521:1, L0375:1, L0651:1,L0805:1, L0776:1, L0526:1, L0783:1, L0789:1, L0666:1, L0664:1, H0701:1,H0723:1, L0352:1, H0547:1, H0658:1, H0670:1, H0648:1, H0651:1, H0436:1,L0740:1, L0754:1, L0752:1, L0757:1, S0436:1, L0591:1, L0592:1 andH0293:1. 53 HE2PO93 771655 63 AR219:19, AR218:19, AR313:13, AR299:13,AR185:13, AR059:11, AR055:10, AR316:10, AR060:10, AR300:9, AR096:8,AR104:7, AR039:7, AR240:6, AR282:6, AR283:5, AR277:3 L0803:5, L0731:5,S0422:4, L2903:3, S0408:2, H0040:2, L0766:2, L0666:2, L2657:2, H0144:2,H0648:2, L0748:2, L0439:2, L0754:2, L0779:2, H0170:1, H0171:1, S0114:1,H0657:1, L2285:1, S0354:1, S0360:1, H0580:1, H0742:1, H0741:1, H0749:1,L2777:1, L0717:1, H0411:1, H0431:1, H0586:1, H0052:1, H0596:1, H0014:1,S0388:1, S0051:1, S0003:1, H0591:1, 50042:1, H0625:1, H0509:1, L0598:1,H0026:1, L0763:1, L0639:1, L0372:1, L0646:1, L0641:1, L0768:1, L0649:1,L0651:1, L0805:1, L0776:1, L0635:1, L0664:1, L0665:1, L2264:1, L2262:1,S0374:1, L0438:1, L0352:1, H0672:1, S0380:1, H0696:1, H0134:1, S0406:1,H0478:1, L0758:1, L0759:1, S0436:1, S0011:1 and S0424:1. 54 HE6FU11827236 64 AR089:8, AR104:6, AR039:6, AR096:5, AR060:5, AR313:5, AR185:5,AR055:4, AR284:4, AR266:4, AR316:4, AR282:4, AR292:4, AR299:4, AR202:4,AR218:3, AR289:3, AR283:3, AR298:3, AR277:3, AR182:3, AR184:3, AR280:3,AR250:3, AR219:3, AR315:3, AR281:3, AR300:3, AR169:3, AR294:3, AR240:3,AR291:3, AR268:2, AR172:2, AR033:2, AR270:2, AR248:2, AR310:2, AR175:2,AR238:2, AR241:2, AR265:2, AR232:2, AR285:2, AR183:2, AR177:2, AR227:2,AR269:2, AR263:2, AR290:2, AR288:2, AR293:2, AR229:2, AR267:2, AR061:2,AR176:2, AR257:2, AR271:2, AR296:2, AR286:2, AR272:2, AR259:1, AR206:1,AR256:1, AR214:1, AR231:1, AR247:1, AR295:1, AR204:1, AR237:1, AR226:1,AR234:1, AR308:1, AR253:1, AR053:1, AR311:1, AR205:1, AR243:1, AR233:1,AR173:1, AR312:1, AR251:1, AR314:1 L0759:2, H0706:1, H0123:1, H0024:1,H0100:1, L07941 and L0789:1. 55 HE6FV29 588454 65 AR219:37, AR218:36,AR315:34, AR280:34, AR271:33, AR244:29, AR089:29, AR314:29, AR243:28,AR281:26, AR282:25, AR273:25, AR205:24, AR192:22, AR206:22, AR198:19,AR247:19, AR316:19, AR039:19, AR231:18, AR269:17, AR246:17, AR204:16,AR234:16, AR299:16, AR313:15, AR194:15, AR055:14, AR186:14, AR237:14,AR060:14, AR241:13, AR270:13, AR293:12, AR240:12, AR232:11, AR238:11,AR251:10, AR300:10, AR061:10, AR233:10, AR227:10, AR291:9, AR185:9,AR202:9, AR266:9, AR226:9, AR229:9, AR184:8, AR179:8, AR182:8, AR175:8,AR312:8, AR268:8, AR289:7, AR284:7, AR249:7, AR183:7, AR310:7, AR267:7,AR052:7, AR033:7, AR296:7, AR290:7, AR265:7, AR177:7, AR309:7, AR292:6,AR298:6, AR275:6, AR277:6, AR285:6, AR294:5, AR248:5, AR053:5, AR253:5,AR295:5, AR286:5, AR259:5, AR274:4, AR258:4, AR213:4, AR096:4, AR256:4,AR104:4, AR283:3, AR263:2 S0440:32, S0476:22, H0494:20, L0754:17,S0372:16, S0132:13, L0666:13, S0330:13, H0046:12, H0586:11, H0587:11,S0328:11, S0360:10, S0436:9, S0356:8, H0622:8, S0003:7, L0806:7,H0648:7, L0747:7, L0752:7, H0674:6, L0777:6, L0362:6, L0662:5, L0659:5,L0601:5, S0430:4, S0358:4, S0408:4, H0592:4, S0214:4, H0039:4, H0031:4,H0551:4, H0264:4, H0S60:4, L0763:4, L0653:4, L5623:4, L0663:4, S0376:3,S0444:3, S0410:3, H0370:3, H0600:3, H0644:3, L0646:3, L0649:3, L0776:3,L0783:3, L0809:3, L0665:3, H0696:3, S0406:3, S3014:3, L0755:3, S0434:3,L0591:3, H0170:2, S134:2, H0662:2, S0442:2, H0393:2, H0596:2, H0597:2,H0688:2, H0553:2, H0032:2, H0169:2, H0598:2, H0090:2, H0379:2, H0380:2,L0770:2, L0372:2, L0549:2, L0376:2, L0517:2, L0518:2, L5622:2, H0658:2,H0670:2, S0380:2, S0152:2, S0350:2, S0027:2, L0744:2, L0779:2, L0759:2,L0599:2, S0196:2, S0456:2, H0171:1, H0556:1, T0002:1, H0713:1, H0483:1,H0663:1, L0005:1, S0354:1, T0008:1, H0742:1, H0741:1, H0411:1, H0549:1,T0039:1, H0013:1, L0021:1, H0349:1, S0010:1, H0204:1, L0738:1, H0545:1,H0014:1, H0015:1, H0373:1, H0355:1, H0510:1, H0615:1, L0483:1, L0142:1,L0143:1, H0166:1, H0673:1, H0708:1, H0591:1, H0038:1, H0040:1, H0634:1,T0067:1, H0272:1, H0487:1, H0412:1, H0623:1, H0059:1, H0100:1, S0352:1,S0382:1, S0448:1, S0306:1, S0438:1, S0472:1, H0646:1, L0503:1, L0640:1,L0637:1, L0761:1, L0772:1, L0764:1, L0771:1, L0648:1, L0794:1, L5564:1,L0551:1, L0805:1, L0382:1, L0519:1, L0789:1, L0532:1, L0664:1, H0144:1,H0520:1, H0547:1, S0126:1, H0689:1, H0711:1, H0435:1, H06591, H06661,S03781, H07041, S0044:1, H0555:1, S0392:1, S0322:1, L0748:1, L0740:1,L0745:1, L0749:1, L0756:1, L0757:1 and S0242:1. 56 HE8TY46 899528 66AR226:12, AR227:11, AR238:10, AR237:10, AR175:9, AR183:9, AR232:9,AR234:8, AR182:8, AR233:7, AR291:7, AR293:7, AR269:7, AR104:7, AR184:7,AR060:6, AR298:6, AR059:6, AR270:6, AR292:6, AR285:6, AR268:6, AR179:6,AR295:5, AR290:5, AR284:5, AR289:5, AR294:5, AR055:5, AR316:5, AR229:5,AR231:5, AR296:5, AR185:5, AR247:5, AR096:5, AR240:5, AR265:5, AR313:5,AR256:5, AR259:4, AR266:4, AR219:4, AR282:4, AR258:4, AR251:4, AR061:4,AR299:4, AR177:4, AR309:4, AR286:4, AR267:4, AR033:4, AR300:3, AR039:3,AR244:3, AR218:3, AR310:3, AR283:3, AR277:3, AR253:3, AR213:2, AR312:2,AR186:2, AR271:1, AR052:1, AR314:1 H0253:8, L0439:8, L0769:7, H0618:6,L0758:6, H0052:5, L0749:5, H0617:4, H0135:4, L0766:4, S0406:4, S0001:3,H0255:3, S0410:3, H0619:3, L3655:3, S0422:3; L0775:3, L0378:3, H0547:3,H0521:3, L0742:3, L0750:3, L0755:3, L0757:3, S0434:3, L0605:3, H0381:2,H0419:2, H0341:2, S0420:2, H0733:2, H0749:2, H0550:2, H0438:2, H0599:2,H0318:2, H0046:2, H0050:2, H0012:2, H0024:2, S0050:2, T0010:2, L0455:2,H0412:2, H0413:2, H0494:2, L0772:2, L0645:2, L0764:2, L0771:2, L0662:2,L0666:2, L0665:2, L0438:2, H0520:2, H0519:2, H0134:2, L0741:2, L0748:2,L0751:2, L0747:2, L0777:2, L0759:2, H0445:2, L0596:2, L0603:2, L0411:1,H0556:1, S0114:1, S0218:1, H0656:1, S0116:1, H0125:1, S0418:1, S0354:1,S0360:1, H0729:1, H0730:1, H0741:1, H0722:1, H0728:1, H0747:1, H0771:1,L0717:1, S0278:1, H0549:1, H0370:1, H0392:1, H0613:1, H0013:1, H0427:1,H0575:1, T0082:1, H0706:1, H0036:1, H0421:1, S0049:1, H0194:1, H0085:1,H0231:1, L0041:1, H0041:1, H0009:1, H0123:1, H0620:1, H0199:1, H0246:1,H0014:1, L0163:1, H0594:1, S6028:1, H0266:1, H0188:1, H0687:1, H0288:1,H0033:1, H0181:1, S0364:1, S0366:1, S0036:1, H0038:1, H0616:1, H0264:1,H0268:1, H0117:1, S0038:1, H0100:1, L0351:1, L0435:1, T0041:1, T0042:1,S0448:1, S0142:1, S0002:1, H0529:1, L0796:1, L0639:1, L3904:1, L5575:1,L3905:1, L5566:1, L0761:1, L0374:1, L0648:1, L0768:1, L0649:1, L0803:1,L0375:1, L0805:1, L0776:1, L0655:1, L0659:1, L0526:1, L0783:1, L5622:1,L0793:1, L0709:1, L3821:1, L2257:1, L2259:1, L0710:1, L2261:1, L2264:1,L2262:1, L2654:1, H0144:1, H0690:1, H0660:1, S0330:1, H0539:1, S0378:1,S0152:1, H0522:1, H0694:1, H0555:1, H0436:1, S3012:1, S0390:1, S3014:1,S0028:1, L0743:1, L0779:1, L0752:1, H0444:1, S0436:1, L0581:1, H0543:1,H0423:1, S0458:1 and H0506:1. 57 HE9EA10 827796 67 L0794:12, H0620:3,L0756:2, L0759:2, S0408:1, S0049:1, H0544:1, H0012:1, H0615:1, H0040:1,L0764:1, L0803:1, L0806:1, L0789:1, H0144:1, H0547:1, L0779:1, L0597:1and L0595:1. 58 HEBCY54 600355 68 AR104:9, AR277:6, AR283:5, AR055:4,AR096:4, AR060:4, AR240:4, AR282:2, AR316:2, AR185:2, AR300:2, AR299:2,AR089:2, AR039:2, AR313:1, AR218:1 L0438:3, L0748:3, T0010:2, L0351:2,L0769:2, H0521:2, L0439:2, L0747:2, S0116:1, S0354:1, S0007:1, H0619:1,H0253:1, H0565:1, H0135:1, L0641:1, L0521:1, L0774:1, L0809:1, L0789:1,H0520:1, L0755:1, L0758:1 and H0445:1. 59 HEBFR46 847064 69 AR313:58,AR039:47, AR300:30, AR096:29, AR299:29, AR277:28, AR089:27, AR185:27,AR316:22, AR219:22, AR104:21, AR218:20, AR240:20, AR282:15, AR060:15,AR055:11, AR283:7 H0457:10, H0550:5, H0436:5, H0549:4, H0616:4, L0519:4,H0556:3, H0580:3, S0007:3, S0046:3, L0809:3, L0747:3, L0777:3, S0436:3,H0295:2, T0040:2, H0266:2, L0761:2, L0783:2, L0789:2, H0658:2, H0521:2,L0753:2, L0731:2, L0596:2, H0543:2, S0040:1, S0116:1, S0282:1, H0662:1,H0402:1, H0125:1, L0534:1, L0562:1, S0356:1, S0358:1, H0749:1, L3816:1,H0559:1, H0069:1, H0599:1, H0618:1, H0253:1, H0581:1, H0546:1, H0123:1,S0051:1, H0083:1, H0687:1, H0284:1, H0124:1, H0038:1, H0551:1, H0623:1,S0038:1, T0041:1, S0440:1, S0150:1, L3818:1, S0002:1, L0763:1, L0769:1,L5575:1, L0627:1, L0800:1, L0662:1, L0803:1, L0793:1, L0666:1, L2264:1,L3825:1, L3827:1, L3828:1, H0547:1, H0519:1, H0539:1, S0037:1, S0206:1,L0748:1, L0749:1, H0595:1, L0593:1, S0194:1 and S0276:1. 60 HEBGE07798096 70 S0007:1 61 HEGAU15 834379 71 AR299:9, AR039:8, AR300:8,AR055:7, AR240:7, AR060:7, AR313;6, AR282:6, AR277:6, AR104:5, AR059:5,AR096:5, AR185:5, AR316:4, AR283:4, AR218:4, AR219:3 H0550:2, L0749:2,H0318:1 and H0555:1. 62 HBTC116 844543 72 AR282:28, AR055:21, AR060:18,AR219:17, AR089:16, AR218:15, AR104:14, AR299:14, AR277:14, AR185:14,AR300:13, AR240:11, AR283:10, AR316:9, AR096:9, AR039:9, AR313:4H0046:6, L0747:6, L0756:6, L0803:5, L0740:5, L0662:4, L0748:4, S0360:3,H0620:3, H0014:3, H0674:3, L0774:3, L5622:3, L0439:3, S0408:2, H0431:2,L0761:2, L0794:2, L0663:2, H0659:2, L0751:2, L0779:2, L0596:2, L0588:2,T0049:1, S0442:1, S0376:1, S0444:1, S0468:1, S0045:1, S0476:1, H0645:1,H0549:1, H0550:1, T0109:1, H0013:1, H0156:1, H0599:1, H0575:1, T0048:1,H0196:1, H0544:1, H0050:1, H0510:1, H0292:1, H0039:1, H0135:1, H0616:1,S0016:1, L0640:1, L0770:1, L0637:1, L3905:1, L0388:1, L0805:1, L0776:1,L0659:1, L0809:1, L0790:1, L0792:1, L0666:1, L0664:1, H0144:1, L0438:1,H0547:1, H0519:1, H0689:1, H0672:1, S0328:1, H0521:1, H0627:1, S3014:1,S0027:1, S0028:1, L0780:1, L07571, L07581, S00261 and H0506:1. 63HFCEI04 692438 73 AR055:7, AR060:7, AR104:6, AR240:6, AR282:6, AR218:5,AR185:5, AR283:5, AR089:4, AR300:4, AR313:3, AR299:3, AR316:3, AR096:3,AR039:3, AR277:3, AR219:2 H0009:3 64 HFCFE20 701985 74 H0052:2, H0560:2,H0529:2, L0470:1, S0212:1, H10305:1, S0420:1, S0356:1, H0550:1, S0222:1,H0497:1, S0010:1, H0251:1, H0009:1, H0024:1, H0083:1, H0290:1, H0379:1,H0264:1, S0150:1, S0426:1, L3905:1, H0520:1, H0547:1, S0044:1, S3014:1,S0434:1, L0581:1, L0604:1, H0136:1, H0423:1 and S0424:1. 65 HFPCZ55840840 75 L0756:6, L0439:4, L0777:4, L0662:3, H0672:3, S0358:2, L0659:2,L0666:2, S0031:2, S0360:1, H0411:1, H0369:1, S0222:1, S0220:1, S0005:1,H0575:1, T0082:1, H0050:1, S6028:1, H0169:1, H0100:1, L0769:1, L0774:1,L0776:1, L0647:1, L0663:1, H0660:1, H0651:1, S0146:1, L0743:1, L07571,L03611 and L0462:1. 66 HFPDR62 839400 76 S0222:2, S0114:1, H0305:1,H0449:1 and T0039:1. 67 HFPDS07 821646 77 AR060:37, AR104:33, AR299:19,AR039:13, AR316:12, AR313:1 1, AR185:11, AR055:10, AR096:10, AR277:9,AR218:8, AR240:7, AR089:7, AR300:6, AR282:5, AR283:4, AR219:2 L0803:24,L0439:13, H0052:5, L0804:5, L0774:5, H0090:4, L0659:4, H0521:4, L0751:4,S0222:3, H0486:3, H0622:3, L0766:3, H0144:3, S0126:3, H0656:2, S0360:2,H0580:2, H0575:2, S0346:2, H0046:2, L0455:2, S0036:2, H0623:2, S0002:2,L0775:2, L0607:2, L0790:2, L0438:2, L0748:2, L0740:2, L0752:2, L0757:2,L0759:2, H0422:2, H0222:1, L3659:1, S0418:1, S0356:1, H0437:1, H0587:1,H0590:1, S0010:1, S0665:1, S0049:1, H0263:1, H0572:1, H0562:1, H0569:1,H0051:1, H0275:1, S6028:1, S0003:1, H0252:1, H0400:1, H0591:1, H0551:1,H0264:1, H0488:1, H0056:1, L0351:1, L0370:1, S0438:1, S0422:1, L0637:1,L0646:1, L0662:1, L0809:1, L0647:1, L0367:1, L0666:1, L0665:1, H0701:1,L3811:1, L3824:1, H0547:1, H0648:1, S0152:1, H0522:1, S0406:1, H0436:1,S0028:1, L0777:1, L0755:1, L0758:1, S0260:1, S0436:1, L0366:1, S0196:1and H0542:1. 68 HFVHW43 570948 78 H0393:1 69 HFXAV37 626595 79 AR313:13,AR039:13, AR300:8, AR299:7, AR277:6, AR096:6, AR185:5, AR059:5, AR218:4,AR104:4, AR316:4, AR282:4, AR060:3, AR240:2, AR055:2, AR219:2, AR283:1S0002:2, S0134:1, S0001:1 and L0589:1. 70 HFXFZ46 600361 80 AR218:9,AR219:9, AR185:2, AR039:2, AR104:1, AR055:1, AR060:1 S0001:1 71 HGBHP91693011 81 AR313:32, AR039:24, AR299:22, AR089:21, AR185:17, AR096:16,AR060:15, AR316:13, AR300:13, AR277:13, AR240:12, AR218:11, AR055:11,AR104:11, AR282:9, AR219:9, AR283:5 H0014:1 72 HHEAK45 765278 82AR277:12, AR283:11, AR313:11, AR316:9, AR282:9, AR089:7, AR240:7,AR104:7, AR299:7, AR039:7, AR218:6, AR096:6, AR055:6, AR300:6, AR185:6,AR060:4, AR219:3 L0758:9, L0748:6, L0747:6, L0779:5, L0750:4, H0556:3,S0440:3, H0658:3 H0656:2, L0770:2, L0769:2, L0804:2, L0774:2, H0144:2,H0648:2, L0439:2, L0749:2, L0596:2, H0265:1, S0444:1, H0318:1, H0597:1,H0050:1, H0024:1, H0135:1, H0090:1, H0038:1, H0616:1, H0494:1, L0065:1,S0422:1, H0529:1, L0637:1, L0764:1, L0768:1, L0794:1, L0387:1, L0803:1,L0805:1, L0809:1, L0788:1, L0790:1, L0664:1, L0438:1, H0555:1, L0780:1,L0731:1, H0444:1, H0542:1, H0543:1 and H0423:1. 73 HHEOW19 886174 83AR169:30, AR089:25, AR207:25, AR308:25, AR214:24, AR263:24, AR165:24,AR264:24, AR164:23, AR161:23, AR168:23, AR222:23, AR171:22, AR283:22,AR166:22, AR162:22, AR311:21, AR163:21, AR096:21, AR223:21, AR213:19,AR104:19, AR316:19, AR219:19, AR218:18, AR217:18, AR312:18, AR212:18,AR225:17, AR309:17, AR282:17, AR313:17, AR039:17, AR272:17, AR299:16,AR216:16, AR060:15, AR172:15, AR274:15, AR083:15, AR170:15, AR240:14,AR055:14, AR185:14, AR195:13, AR277:13, AR197:13, AR235:13, AR295:12,AR192:12, AR224:12, AR296:11, AR297:11, AR246:11, AR285:10, AR198:10,AR245:10, AR293:10, AR252:10, AR288:10, AR205:10, AR300:10, AR221:9,AR242:9, AR287:9, AR201:9, AR247:9, AR253:9, AR033:9, AR266:9, AR215:9,AR275:8, AR291:8, AR174:8, AR261:8, AR193:8, AR243:8, AR271:8, AR177:8,AR270:8, AR254:8, AR289:7, AR236:7, AR286:7, AR175:6, AR204:6, AR269:6,AR189:6, AR294:6, ARI8O:6, AR178:5, AR250:5, AR183:5, AR199:5, AR257:5,AR181:5, AR179:5, AR268:5, AR262:5, AR173:5, AR290:5, AR258:5, AR255:4,AR061:4, AR210:4, AR191:4, AR190:4, AR229:4, AR196:4, AR176:4, AR188:3,AR226:3, AR239:3, AR182:3, AR200:3, AR234:3, AR238:3, AR267:3, AR232:3,AR230:3, AR203:3, AR256:3, AR231:3, AR211:3, AR237:3, AR233:2, AR227:2,AR260:2, AR228:1 L0748:4, L0745:4, L0775:3, L0776:3, L0758:3, H0458:2,H0050:2, S0003:2, H0529:2, L0747:2, L0599:2, L0362:2, H05561, S0116:1,S0282:1, H0662:1, H0305:1, S0420:1, S0444:1, H0329:1, H0345:1, H0411:1,S0278:1, H0438:1, T0039:1, H0635:1, H0156:1, H0235:1, H0327:1, L0471:1,H0428:1, H0031:1, H0644:1, H0032:1, S0366:1, H0038:1, H0616:1, T0067:1,H0477:1, H0059:1, H0560:1, H0625:1, S0422:1, L0769:1, L0761:1, L0667:1,L0771:1, L0662:1, L0806:1, L0655:1, L0809:1, L5622:1, L0789:1, L0790:1,L0665:1, S0052:1, H0144:1, H0520:1, H0547:1, H0519:1, H0435:1, H0539:1,S0044:1, S0392:1, L0754:1, L0749:1, L0750:1, L0779:1, L0755:1, L0759:1,S0434:1, L0608:1, H0543:1 and S0452:1. 74 HHFFS40 824059 84 AR219:22,AR277:18, AR283:17, AR218:16, AR039:15, AR282:15, AR089:14, AR316:13,AR313:13, AR096:12, AR299:12, AR104:12, AR240:10, AR055:10, AR300:10,AR185:9, AR060:8 S0422:7, L0748:6, L0591:6, L0766:5, L0754:5, H0423:5,S0408:4, H0069:4, L0803:4, L0602:4, H0657:3, S0442:3, S0046:3, H0596:3,S0003:3, H0032:3, H0169:3, H0674:3, L0662:3, L0794:3, L0526:3, H0670:3,L0740:3, L0759:3, S0134:2, S0212:2, H0661:2. S0444:2, H0046:2, L0471:2,H0355:2, H0038:2, H0100:2, L0564:2, S0440:2, H0529:2, L0770:2, L0769:2,L0667:2, L0771:2, L0521:2, L0804:2, L0805:2, L0384:2, L0809:2, L0665:2,H0659:2, L0743:2, L0750:2, L0731:2, S0436:2, L0592:2, L0599:2, L0608:2,L0362:2, H0171:1, H0556:1, H0686:1, H0713:1, H0717:1, H0738:1, H0740:1,H0656:1, H0663:1, H0662:1, H0402:1, S0356:1, H0742:1, H0730:1, H0747:1,S0222:1, H0574:1, H0632:1, H0486:1, H0013:1, H0581:1, S0049:1, H0052:1,H0194:1, H0309:1, H0263:1, H0123:1, H0050:1, H0373:1, H0510:1, 56028:1,H0266:1, H0615:1, L0483:1, H0644:1, L0143:1, H0708:1, H0135:1, H0163:1,H0090:1, H0616:1, T0067:1, H0488:1, H0412:1, H0059:1, H0494:1, S0382:1,S0306:1, S0450:1, H0509:1, H0641:1, H0647:1, H0646:1, L0520:1, L0763:1,L0637:1, L0373:1, L0363:1, L5564:1, L0775:1, L0375:1, L0651:1, L0655:1,L0661:1, L0527:1, L0656:1, L0659:1, L0518:1, L0532:1, L0663:1, L0664:1,S0374:1, H0682:1, H0658:1, H0660:1, H0672:1, H0539:1, H0521:1, S0044:1,S0406:1, H0478:1, L0744:1, L0439:1, L0747:1, L0779:1, L0777:1, L0758:1,L0480:1, L0595:1, H0667:1, S0192:1, S0194:1, S0196:1, H0422:1 andS0424: 1. 75 HHGCS78 634605 85 AR277:76, AR283:71, AR219:57, AR218:56,AR316:56, AR059:52, AR313:52, AR240:51, AR055:45, AR282:45, AR299:44,AR104:41, AR096:41, AR185:34, AR039:33, AR060:31, AR300:30 L0770:7,H0333:3, L0783:2, L0731:2, H0445:2, S0418:1, H0741: 1, S0002:1, L0369:1,L0643:1, L0764:1, L0794:1, L0803:1, L0775:1, L0375:1, L0378:1, L0655:1,L0509:1, L0666:1, L0664:1, L0754:1, L0747:1, L0749:1, L0752:1 andL0591:1. 76 HHPSA85 658695 86 AR104:25, AR313:9, AR055:5, AR060:5,AR240:5, AR096:5, AR277:5, AR282:4, AR089:4, AR316:4, AR185:4, AR218:4,AR300:4, AR299:3, AR039:3, AR283:2, AR219:2 L0756:5, H0051:4, L0438:4,L0759:4, S0031:4, S0007:3, S6028:3, L0666:3, L0439:3, H0556:2, S6024:2,S0300:2, H0013:2, S0036:2, L0770:2, L0411:1, L0393:1, H0393:1, L3653:1,L3657:1, H0581:1, H0235:1, H0327:1, H0046:1, H0009:1, L0157:1, H0201:1,S0051:1, H0399:1, H0064:1, H0038:1, H0040:1, H0634:1, H0100:1, L0638:1,L0796:1, L0768:1, L0794:1, L0766:1, L0803:1, L06061, L07911, L07921,H0144:1, H06981, L38111, H05471, H0519:1, H0659:1, L0779:1, L0752:1,S0260:1 and H0136:1. 77 HHSBI06 639097 87 AR218:14, AR060:11, AR282:11,AR055:11, AR089:10, AR219:10, AR185:10, AR277:10, AR039:9, AR104:9,AR299:8, AR316:8, AR300:8, AR313:8, AR283:7, AR096:7, AR240:5 L0766:12,L0794:7, L0439:7, L0749:7, L0803:6, L0740:6, L0745:6, H0052:5, L0754:5,L3181:4, L0770:4, L0666:4, L0748:4, H0553:3, L0790:3, L0589:3, H0543:3,S0114:2, S0134:2, H0650:2, S0354:2, S0444:2, H0747:2, S0476:2, H0393:2,H0549:2, H0586:2, H0013:2, H0599:2, H0014:2, S0051:2, S0003:2, H0032:2,H0674:2, H0135:2, S0142:2, L0372:2, L0800:2, L0764:2, L0805:2, L0655:2,L0657:2, L0659:2, L0809:2, L0789:2, L0792:2, H0144:2, L0438:2, H0684:2,H0658:2, H0539:2, H0521:2, S0406:2, S0028:2, L0750:2, L0779:2, L0777:2,L0752:2, L0731:2, L0758:2, S0436:2, H0653:2, H0542:2, H0556:1, H0716:1,H0381:1, S0116:1, H0661:1, S0356:1, S0442:1, S0360:1, H0675:1, H0734:1,L2255:1, L3726:1, H0261:1, S0222:1, L3499:1, T0114:1, H0706:1, H0036:1,H0318:1, H0581:1, L0738:1, H0123:1, H0620:1, S0050:1, H0015:1, H0051:1,H0355:1, H0416:1, H0286:1, H0328:1, H0428:1, H0622:1, T0006:1, H0030:1,H0031:1, H0644:1, H0617:1, L0055:1, H0124:1, H0163:1, H0038:1, H0040:1,H0616:1, H0551:1, H0264:1, H0102:1, S0112:1, L0564:1, H0280:1, H0494:1,H0561:1, S0440:1, H0633:1, L3815:1, S0002:1, L0763:1, L0769:1, L0761:1,L0646:1, L0642:1, L0644:1, L0645:1, L0648:1, L0662:1, L0363:1, L0775:1,L0375:1, L0651:1, L0784:1, L0806:1, L0653:1, L0807:1, L0658:1, L0540:1,L5622:1, L0368:1, L0665:1, L2655:1, L2257:1, L2263:1, L2258:1, L2262:1,S0374:1, H0723:1, L3811:1, L2670:1, H0547:1, L3215:1, H0648:1, H0672:1,S0328:1, H0753:1, H0522:1, H0436:1, S0392:1, H06261, L0759:1, S0031:1,H0445:1, S0434:1, L0596:1, L0588:1, S0192:1, H0423:1, S0424:1 andH0352:1. 78 HHSBI65 801910 88 AR176:10, AR216:9, AR217:8, AR168:8,AR169:8, AR182:8, AR16L:8, AR196:8, AR162:8, AR214:8, AR228:8, AR269:8,AR231:8, AR233:7, AR171:7, AR207:7, AR229:7, AR181:7, AR223:7, AR163:7,AR198:7, AR165:7, AR172:7, AR225:7, AR267:7, AR224:7, AR266:6, AR268:6,AR170:6, AR164:6, AR237:6, AR221:6, AR222:6, AR177:6, AR179:6, AR235:6,AR270:6, AR183:6, AR204:6, AR288:6, AR053:6, AR239:6, AR193:5, AR236:5,AR250:5, AR191:5, AR264:5, AR293:5, AR296:5, AR055:5, AR238:5, AR247:5,AR309:5, AR300:5, AR178:5, AR295:5, AR290:5, AR294:5, AR060:5, AR061:5,AR287:5, AR257:5, AR201:5, AR282:5, AR291:5, AR175:5, AR311:4, AR261:4,AR234:4, AR289:4, AR275:4, AR262:4, AR252:4, AR242:4, AR213:4, AR253:4,AR297:4, AR203:4, AR277:4, AR180:4, AR212:4, AR200:4, AR316:4, AR286:4,AR274:4, AR255:4, AR312:4, AR240:4, AR174:4, AR215:4, AR039:4, AR192:4,AR263:4, AR205:4, AR283:3, AR232:3, AR271:3, AR285:3, AR190:3, AR226:3,AR185:3, AR033:3, AR246:3, AR230:3, AR188:3, AR308:3, AR227:3, AR096:3,AR173:3, AR313:3, AR089:3, AR195:3, AR272:3, AR199:3, AR189:3, AR260:3,AR197:3, AR299:3, AR104:2, AR210:2, AR258:2, AR211:2, AR256:2, AR243:2,AR218:2, AR219:2 L0439:7, L0794:5, L0766:5, S0354:2, H0549:2, S0051:2,S0142:2, L0372:2, L0809:2, L0438:2, H0658:2, H0650:1, H0381:1, S0116:1,S0356:1, S0360:1, H0261:1, H0586:1, H0486:1, H0036:1, H0052:1, L0738:1,H0457:1, H0014:1, H0051:1, H0617:1, H0032:1, H0561:1, S0440:1, H0633:1,L0763:1, L0761:1, L0800:1, L0644:1, L0645:1, L0764:1, L0648:1, L0655:1,L0657:1, L0658:1, L0368:1, L0665:l, L3811:1, S0044:1, S0406:1, H0626:1,L0731:1, S0434:1, S0436:1, H0653:l and H0423:1. 79 HHSGL28 801912 89L0439:8, L0438:3, S0440:2, L0666:2, H0170:1, S0442:1, H0318:1, S0049:1,H0052:1, H0050:1, H0057:1, S0388:1, S0214:1, H0598:1, S0036:1. H0063:1,H0551:1, L0520:1, L0796:1, L0662:1, L0766:1, L0664:1, H0547:1, H0435:1,H0521:1, L0779:1, L0777:1, L0752:1 and L0594:1. 80 HISAT67 843549 90AR283:20, AR282:15, AR277:11, AR089:11, AR219:11, AR299:10, AR218:10,AR316:9, AR313:8, AR096:8, AR185:8, AR104:7, AR240:7, AR055:6, ARO6O:6,AR039:6, AR300:6 L0751:8, L0754:6, L0731:6, H0556:5, L0766:5, L0439:5,L0750:5, L0770:4, L0666:4, H0521:4, S0356:3, H0052:3, H0424:3, L0776:3,S0406:3, S0418:2, S0442:2, H0580:2, H0733:2, H0486:2, H0575:2, S0438:2,L0769:2, L3905:2, L0659:2, L0663:2, L0665:2, L0748:2, L0749:2, S0436:2,T0002:1, H0159:1, S6024:1, S0134:1, H0656:1, H0484:1, S0358:1, S0360:1,H0742:1, S0046:1, H0393:1, S0278:1, H0607:1, H0586:1, H0642:1, H0632:1,H0427:1, L0021:1, H0599:1, H0318:1, H0746:1, H0194:1, L0738:1, H0178:1,H0566:1, H0051:1, T0010:1, H0408:1, H0290:1, H0328:1, H0401:1, H0417:1,H0553:1, H0617:1, H0040:1, H0264:1, H0623:1, H0059:1, T0041:1, H0494:1,H0561:1, H0509:1, S0144:1, L0763:1, L0638:1, L5565:1, L0772:1, L0373:1,L0764:1, L0662:1, L0626:1, L0363:1, L0649:1, L0650:1, L0774:1, L0806:1,L0654:1, L0789:1, L0664:1, L3822:1, H0699:1, S0374:1, L3828:1, H0547:1,H0689:1, H0659:1, H0658:1, H0670:1, H0672:1, H0539:1, L0740:1, L0746:1,L0752:1, L0755:1, L0757:1, L0584:1, L0596:1, L0608:1 and H0352:1. 81HJBCU75 638329 91 AR162:12, AR161:12, AR163:11, AR186:10, AR244:10,AR273:8, AR222:8, AR225:8, AR221:8, AR214:8, AR216:8, AR224:7, AR223:7,AR282:7, AR215:7, AR052:7, AR202:7, AR200:6, AR206:6, AR176:6, AR269:6,AR235:6, AR272:6, AR217:6, AR055:6, AR182:6, AR264:6, AR275:6, AR061:6,AR183:5, AR168:5, AR171:5, AR309:5, AR270:5, AR228:5, AR290:5, AR268:5,AR310:5, ARL8L:5, AR257:5, AR060:5, AR255:5, AR165:5, AR191:5, AR164:5,AR274:5, AR247:5, AR246:4, AR166:4, AR178:4, AR311:4, AR291:4, AR312:4,AR236:4, AR173:4, AR249:4, AR233:4, AR240:4, AR288:4, AR174:4, AR267:4,AR239:4, AR204:4, AR185:4, AR287:4, AR172:4, AR192:4, AR297:4, AR213:4,AR194:4, AR177:4, AR294:4, AR261:4, AR184:4, AR190:4, AR170:3, AR284:3,AR262:3, AR210:3, AR188:3, AR089:3, AR313:3, AR196:3, AR298:3, AR266:3,AR175:3, AR033:3, AR260:3, AR285:3, AR189:3, AR316:3, AR231:3, AR251:3,AR293:3, AR296:3, AR237:3, AR286:3, AR265:3, AR243:3, AR277:3, AR300:3,AR039:3, AR289:3, AR315:3, AR179:3, AR271:3, AR234:3, AR263:3, AR199:3,AR283:3, AR203:3, AR096:3, AR299:3, AR295:3, AR229:3, AR230:3, AR292:3,AR180:3, AR104:2, AR198:2, AR308:2, AR219:2, AR053:2, AR218:2, AR211:2,AR205:2, AR238:2, AR241:2, AR258:2, AR256:2, AR226:2, AR232:2, AR280:2,AR169:1, AR227:1, AR259:1, AR201:1 L0805:3, H0556:2, H0046:2, S0022:2,L0764:2, L0662:2, L0748:2, H0013:1, H0050:1, H0039:1, H0040:1, H0087:1,T0042:1, L0643:1, L0794:1, L0803:1, L0804:1, L0807:1, L0809:1, L0666:1,H0144:1, L0749:1, L0779:1 and L0758:1. 82 HJMAA03 824062 92 AR207:12,AR309:11, AR192:11, AR252:10, AR053:9, AR212:9, AR242:9, AR235:9,AR213:8, AR215:8, AR198:8, AR170:8, AR169:8, AR161:8, AR162:8, AR253:8,AR223:8, AR165:8, AR166:8, AR263:7, AR163:7, AR164:7, AR274:7, AR224:7,AR245:7, AR264:7, AR214:7, AR195:7, AR217:7, AR174:7, AR197:7, AR261:7,AR311:7, AR221:7, AR282:6, AR308:6, AR222:6, AR240:6, AR312:6, AR205:6,AR171:6, AR168:6, AR193:6, AR313:6, AR246:6, AR177:6, AR173:6, AR277:6,AR216:6, AR247:6, AR180:6, AR225:6, AR283:5, AR269:5, AR300:5, AR089:5,AR201:5, AR272:5, AR297:5, AR189:5, AR204:5, AR183:5, AR299:5, AR175:5,AR288:5, AR176:5, AR295:5, AR271:5, AR250:5, AR096:5, AR275:5, AR270:4,AR316:4, AR196:4, AR191:4, AR286:4, AR178:4, AR290:4, AR185:4, AR268:4,AR296:4, AR291:4, AR257:4, AR033:4, AR199:4, AR181:4, AR039:4, AR236:4,AR229:4, AR243:4, AR285:4, AR254:4, AR289:4, AR238:3, AR172:3, AR293:3,AR262:3, AR190:3, AR287:3, AR179:3, AR200:3, AR055:3, AR104:3, AR060:3,AR188:3, AR239:3, AR182:3, AR233:3, AR258:3, AR294:3, AR061:3, AR237:3,AR231:3, AR234:3, AR226:3, AR203:3, AR255:3, AR232:3, AR230:2, AR211:2,AR227:2, AR228:2, AR267:2, AR210:2, AR266:2, AR219:2, AR260:1, AR218:1,AR256:1 L0749:8, L0803:5, 1.0748:5, L0777:5, L0794:4, L0766:4, L0804:4,H0135:3, H0551:3, L0754:3, L0599:3, H0542:3, H0556:2, H0545:2, H0674:2,L0764:2, L0774:2, L0776:2, L0655:2, H0521:2, L0439:2, L0752:2, L0731:2,L0596:2, H0395:1, H0713:1, H0483:1, H0663:1, S0358:1, H0580:1, H0329:1,S0045:1, H0453:1, H0427:1, H0599:1, H0706:1, H0150:1, H0123:1, L0471:1,L0163:1, H0051:1, H0275:1, S0003:1, S0214:1, H0628:1, H0090:1, H0040:1,H0087:1, T0067:1, H0412:1, H0494:1, H0509:1, H0633:1, H0647:1, S0344:1,L0769:1, L0637:1, L0761:1, L0772:1, L0800:1, L0374:1, L0771:1, L0363:1,L0768:1, L0806:1, L0659:1, L0382:1, L0809:1, L0545:1, L0789:1, L0666:1,H0519:1, H0659:1, S0152:1, S0404:1, L0751:1, L0747:1, L0750:1, L0779:1,S0436:1, L0608:1, S0276:1, H0543:1, H0506:1 and H0352:1. 83 EKABU43838573 93 AR219:2, AR282:1, AR300:1, AR316:1 L0794:7, L0803:3, H0052:2,S0250:2, H0032:2, H0494:2, H0529:2, L0666:2, L0663:2, L0747:2, L0759:2,H0657:1, H0664:1, H0662:1, S0442:1, H0741:1, H0735:1, H0733:1, S0046:1,H0640:1, H0331:1, H0559:1, T0039:1, H0013:1, S0280:1, H0318:1, T0110:1,H0024:1, S0364:1, H0591:1, H0038:1, H0040:1, S0142:1, L0640:1, L0667:1,L0764:1, L0662:1, L0804:1, L0659:1, L0517:1, L0789:1, L4559:1, L0664:1,S0126:1, H0435:1, H0539:1, S0152:1, H0521:1, H0522:1, S0027:1, L0779:1,L0758:1, L0485:1, L0601:1, S0026:1, H0667:1, S0192:1, H0542:1 andH0506:1. 84 HKACI79 853361 94 AR313:63, AR039:48, AR300:33, AR096:31,AR089:31, AR277:26, AR185:25, AR299:24, AR316:20, AR240:19, AR218:15,AR219:14, AR282:12, AR104:12, AR060:9, AR055:6, AR283:3 H0659:2,S0418:1, L0004:1, H0041:1, H0087:1, H0494:1, H0646:1, S0422:1, L0373:1,L07661, L06651, S0380:1, L0748:1, L0740:1 and L0589:1. 85 HKIXC44 71621395 AR104:28, AR055:19, AR240:15, AR219:11, AR218:11, AR185:10, AR060:9,AR299:7, AR089:7, AR283:7, AR282:6, AR096:5, AR316:5, AR300:5, AR313:5,AR039:4, AR277:3 L0770:7, L0742:5, L0439:4, L0776:3, S0358:2, H0619:2,S0222:2, L0769:2, L0638:2, L0796:2, L0805:2, H0593:2, L0753:2, L0485:2,L0608:2, H0329:1, H0351:1, H0441:1, H0611:1, H0371:1, H0013:1, H0196:1,H0052:1, H02511, H00411, H00241, H06221, S0366:1, H0623:1, L0648:1,L05231, L08061, L0788:1, L0666:1, L0663:1, H0648:1, H0539l, S0152:1,L0612:1, L0777:1, L0599:1 and S0242:1. 86 HKTAB41 695732 96 AR277:83,AR283:74, AR219:65, AR313:56, AR316:50, AR089:49, AR218:47, AR282:46,AR104:45, AR055:42, AR185:41, AR299:40, AR096:35, AR039:31, AR240:31,AR060:26, AR300:25 L0794:5, H0574:1 and H0239:1. 87 HLDQU79 740755 97AR253:8, AR171:7, AR245:6, AR243:5, AR183:5, AR263:5, AR264:4, AR250:4,AR269:4, AR060:4, AR180:4, AR270:4, AR309:4, AR162:4, AR268:4, AR161:4,AR165:4, AR192:4, AR176:4, AR164:4, AR055:4, AR163:4, AR213:4, AR195:4,AR271:4, AR166:3, AR275:3, AR240:3, AR282:3, AR312:3, AR246:3, AR178:3,AR181:3, AR311:3, AR168:3, AR289:3, AR182:3, AR193:3, AR217:3, AR179:3,AR212:3, AR237:3, AR238:3, AR299:3, AR199:3, AR252:3, AR229:3, AR242:2,AR185:2, AR300:2, AR277:2, AR175:2, AR293:2, AR257:2, AR308:2, AR177:2,AR198:2, ARO61:2, AR214:2, AR174:2, AR104:2, AR231:2, AR316:2, AR201:2,AR233:2, AR230:2, AR224:2, AR236:2, AR239:2, AR228:2, AR188:2, AR223:2,AR189:2, AR247:2, AR294:2, AR226:2, AR266:2, AR221:2, AR285:2, AR191:2,AR089:2, AR216:2, AR200:2, AR207:2, AR272:2, AR232:2, AR190:2, AR290:2,AR283:2, AR096:2, AR222:2, AR296:2, AR039:2, AR267:2, AR205:2, AR211:1,AR196:1, AR173:1, AR033:1, AR218:1, AR295:1, AR255:1, AR262:1, AR215:1,AR227:1, AR254:1, AR234:1, AR313:1, AR203:1, AR256:1, AR169:1, AR225:1,AR210:1, AR170:1 L0748:9, L0731:7, L0771:6, L0759:6, H0013:5, L0764:4,L0747:4, L0758:4, H0265:3, H0039:3, H0038:3, L0769:3, L0766:3, L0775:3,H0144:3, L0755:3, S0444:2, S0476:2, H0318:2, H0050:2, L0471:2, H0266:2,L0374:2, L0649:2, L0805:2, L0663:2, L0664:2, H0547:2, S0126:2, H0670:2,L0740:2, L0754:2, L0750:2, L0593:2, H0667:2, H0170:1, H0171:1, H0685:1,H0662:1, S0354:1, S0360:1, H0580:1, H0728:1, H0151:1, H0747:1, L3388:1,H0357:1, H0586:1, H0331:1, H0574:1, H0635:1, H0575:1, H0263:1, H0596:1,H0545:1, H0012:1, H0620:1, H0350:1, H0355:1, H0510:1, H0428:1, H0604:1,H0031:1, H0553:1, S0366:1, H0040:1, H0063:1, H0059:1, H0560:1, H0561:1,S0440:1, S0422:1, H0529:1, L0640:1, L0637:1, L0761:1, L0772:1, L0646:1,L4556:1, L0774:1, L0375:1, L0653:1, L0382:1, L5622:1, L0793:1, L4501:1,H0723:1, L0352:1, S0152:1, S0350:1, H0521:1, H0696:1, S0044:1, H0627:1,S0027:1, L0749:1, L0752:1, H0595:1, S0436:1, L0591:1, L0595:1, L0361:1,S0011:1, S0194:1, S0276:1 and H0423:1. HLDQU79 837599 191 88 HLHAP05638476 98 L0005:3, H0024:2, H0209:1 and H0445:1. 89 HLHCS23 560663 99AR055:5, AR060:4, AR185:3, AR218:3, AR240:3, AR300:3, AR282:3, AR299:2,AR039:2, AR283:2, AR089:2, AR219:2, AR316:2, AR104:2, AR096:1, AR277:1H0024:1 90 HLICE88 840321 100 AR185:21, AR240:19, AR104:13, AR039:13,AR060:13, AR089:13, AR300:12, AR282:11, AR096:11, AR055:10, AR316:10,AR219:10, AR218:9, AR299:7, AR283:7, AR313:7, AR277:4 H0014:72,L3388:60, H0509:49, L0581:44, H0355:43, H0574:32, H0393:30, H0632:21,H0510:18, S0438:18, H0098:15, H0144:14, H0331:13, H0015:8, L0748:8,H0722:7, L3387:7, H0741:5, H0013:5, H0147:4, T0078:4, L0615:3, H0357:3,S0440:3, H0730:2, H0349:2, H0350:2, H0057:2, H0644:2, H0647:2, L0605:2,L0599:2, H0170:1, L0448:1, H0149:1, L0393:1, S0444:1, L3645:1, H0749:1,L2255:1, H0351:1, H0642:1, H0427:1, H0003:1, H0575:1, H0199:1, H0040:1,H07451, L07871, L07471 and S0436:1. 91 HLMGP50 647603 101 AR055:5,AR313:4, AR282:4, AR104:4, AR277:4, AR300:4, AR299:3, AR060:3, AR039:3,AR096:3, AR316:3, AR283:2, AR185:2, AR218:2, AR240:2, AR089:2 H0255:2,H0385:1, L0753:1 and H0595:1. 92 HLMMX62 688051 102 AR060:7, AR055:7,AR313:7, AR299:7, AR089:7, AR218:6, AR185:6, AR240:6, AR039:5, AR300:5,AR277:5, AR096:4, AR219:4, AR283:4, AR104:4, AR316:4, AR282:3 H0255:2,S0410:2, H0052:1 and H0673:1. 93 HLQCX36 584786 103 AR313:27, AR226:23,AR039:23, AR251:21, AR089:17, AR238:15, AR241:14, AR096:14, AR185:14,AR299:14, AR104:13, AR258:13, AR300:13, AR316:12, AR293:12, AR253:12,AR240:12, AR198:11, AR060:11, AR219:11, AR248:11, AR249:10, AR218:10,AR237:10, AR231:10, AR232:10, AR282:10, AR227:10, AR275:10, AR192:10,AR186:9, AR234:9, AR229:8, AR271:8, AR312:8, AR277:8, AR204:8, AR274:7,AR292:7, AR243:7, AR294:7, AR233:7, AR244:7, AR177:7, AR183:7, AR055:7,AR259:6, AR053:6, AR175:6, AR033:6, AR052:6, AR269:6, AR273:5, AR202:5,AR247:5, AR206:5, AR061:5, AR309:5, AR267:5, AR213:5, AR265:5, AR256:4,AR295:4, AR184:4, AR205:4, AR179:4, AR283:3, AR268:3, AR182:3, AR246:3,AR270:3, AR310:3, AR296:2, AR298:2, AR281:2, AR290:2, AR286:2, AR263:2,AR315:2, AR266:2, AR284:1, AR280:1, AR289:1, AR291:1, AR285:1 L0459:1and H0574:1. 94 HLWAF06 658701 104 AR277:1 L0664:2, L0438:2, L0439:2,L0751:2, L0755:2, H0553:1, S0002:1, L0775:1 and L0752:1. 95 HLWDB73838453 105 L0777:13, L0803:9, L0748:9, L0731:6, H0423:5, L0794:4,L0766:4, L0740:4, L0754:4, L0779:4, H0171:3, S0408:3, H0580:3, S0003:3,H0553:3, H0616:3, L0804:3, H0519:3, L0439:3, L0751:3, S0026:3, H0422:3,H0170:2, H0717:2, S0356:2, H0486:2, H0318:2, H10644:2, H0068:2, H0038:2,H0551:2, H0413:2, L0598:2, L0646:2, L0662:2, L0388:2, L0784:2, L0805:2,L0776:2, L0790:2, H0547:2, H0710:2, H0521:2, L0745:2, L0747:2, L0756:2,L0752:2, H0624:1, S0342:1, SO114:1, S0134:1, H0650:1, L0808:1, S0354:1,S0444:1, S0360:1, S0046:1, H0411:1, H0438:1, H0600:1, H0632:1, T0039:1,H0013:1, H0427:1, H0581:1, H0421:1, H0544:1, H0046:1, H0457:1, H0150:1,H0563:1, H0123:1, H0019:1, L0471:1, H0024:1, H0271:1, H0416:1, H0428:1,H0039:1, L0055:1, H0361:1, H0598:1, H0090:1, H0591:1, H0268:1, H0623:1,H0560:1, S0440:1, H0647:1, H0646:1, S0344:1, UNKWN:1, L0763:1, L0770:1,L0769:1, L0637:1, L0667:1, L0764:1, L0767:1, L0768:1, L0649:1, L0775:1,L0375:1, L0806:1, L0807:1, L0659:1, L0783:1, L0791:1, L0792:1, L0663:1,L0664:1, H0144:1, S0374:1, L0438:1, H0520:1, S0126:1, H0658:1, H0648:1,S0330:1, S0152:1, H0696:1, S0406:1, H0576:1, S0028:1, L0749:1, L0780:1,L0755:1, L0758:1, S0031:1, H0595:1, L0596:1, L0581:1, L0604:1, H0665:1and S0194:1. 96 HLYDF73 566869 106 AR277:12, AR283:9, AR282:6, AR316:6,AR300:5, AR055:5, AR089:5, AR104:4, AR299:4, AR185:4, AR096:4, AR218:4,AR313:4, AR240:4, AR039:3, AR219:3, AR060:2 H0445:1 97 HLYGB19 838083107 AR240:33, AR096:27, AR313:22, AR055:15, AR282:15, AR316:13,AR060:10, AR089:9, AR039:9, AR300:7, AR277:7, AR299:7, AR104:5, AR219:4,AR185:4, AR283:4, AR218:1 L0752:10, L0471:9, L0731:8, H0422:8, H0040:5,L0641:5, L0662:4, L0439:4, L0755:4, S0114:3, S0360:3, L0766:3, L0747:3,L0749:3, L0757:3, H0445:3, H0543:3, H0265:2, H0556:2, S0116:2, H0013:2,H0244:2, H0135:2, H0264:2, L0769:2, L0639:2, L0761:2, L0774:2, L0775:2,L0776:2, L0384:2, L0663:2, L0665:2, L0565:2, H0658:2, H0539:2, L3832:2,L0744:2, L0748:2, L0750:2, L0779:2, L0758:2, L0759:2, S0134:1, H0657:1,S0212:1, S0400:1, S0420:1, L3645:1, S0046:1, S0476:1, L0717:1, S0220:1,L2491:1, H0599:1, H0706:1, L0563:1, H0545:1, H0150:1, H0009:1, H0024:1,T0010:1, H0354:1, H0028:1, H0553:1, L0456:1, H0616:1, H0413:1, L0351:1,S0438:1, H0646:1, L3818:1, S0208:1, L0796:1, L3904:1, L0667:1, L0644:1,L0764:1, L0768:1, L0649:1, L0655:1, L0606:1, L0634:1, L0659:1, L0809:1,L0367:1, L0793:1, L0666:1, H0144:1, L0438:1, L2670:1, H0689:1, H0666:1,H0672:1, S0378:1, H0436:1, L0756:1, L0599:1, L0595:1, S0242:1, H0542:1,H0423:1 and L3796:1. 98 HLYGY91 658703 108 AR313:6, AR316:5, AR218:3,AR300:3, AR299:3, AR055:3, AR185:2, AR039:2, AR096:2, A2R277:2, AR219:1,AR089:1 H0692:10, L0777:10, L0805:5, L0803:3, L2497:2, H0328:2, L0662:2,L0794:2, L0809:2, L3832:2, L0748:2, L0752:2, L0599:2, H0170:1, H0402:1,S0444:1, S0360:1, H0747:1, L2486:1, L3503:1, H0427:1, H0644:1, H0038:1,L0800:1, L0648:1, L0804:1, H0670:1, H0478:1, L0731:1, L0758:1, H0445:1,S0434:1, L0591:1 and L0362:1. 99 HMDAB29 584789 109 AR313:127, AR039:86,AR299:64, AR089:56, AR185:51, AR096:50, AR277:50, AR300:42, AR316:37,AR240:33, AR218:27, AR219:25, AR104:22, AR060:22, AR282:20, AR055:16,AR283:9 H0346:1, H0598:1 and S0330:1. 100 HMDAD44 566854 110 AR277:44,AR283:35, AR219:28, AR316:26, AR089:24, AR218:23, AR313:22, AR282:22,AR055:22, AR104:21, AR299:20, AR185:19, AR240:19, AR096:17, AR039:16,AR060:14, AR300:14 L0749:3, H0346:1, H0370:1, H0427:1 and L0439:1. 101HMEDI90 840077 111 AR104:7, AR316:6, AR055:5, AR060:5, AR300:4, AR185:4,AR218:4, AR282:3, AR283:3, AR240:3, AR089:3, AR219:2, AR299:2, AR039:1,AR313:1, AR096:1, AR277:1 L0439:8, S6028:2, H0266:2, L0438:2, L0745:2,L0717:1, S0222:1, H0052:1, H0194:1, H0009:1, T0010:1, S0036:1, L0776:1,L0789:1, S0028:1, L0756:1 and L0779:1. 102 HMIBF07 603528 112 AR055:5,AR060:4, AR240:4, AR300:3, AR299:3, AR104:3, AR283:3, AR219:2, AR218:2,AR185:2, AR039:2, AR089:2, AR277:2, AR096:2, AR316:2, AR282:1, AR313:1S6028:1 103 HMICP65 847403 113 AR313:25, AR039:24, AR277:13, AR104:13,AR300:11, AR096:11, AR299:10, AR185:10, AR089:9, AR219:9, AR316:8,AR218:8, AR240:6, AR060:6, AR282:5, AR055:3, AR283:2 S0474:12, H0156:5,H0650:3, L0666:3, H0341:2, H0393:2, H0486:2, H0052:2, H0039:2, H0135:2,S0330:2, L0748:2, L0439:2, L0757:2, L0601:2, H0224:1, H0225:1, S0134:1,H0583:1, H0657:1, S0212:1, S0282:1, H0735:1, S0046:1, H0550:1, H0431:1,L3653:1, H0013:1, H0042:1, H0590:1, S0010:1, H0318:1, H0046:1, H0009:1,H0050:1, H0242:1, S0388:1, S6028:1, H0271:1, H0031:1, H0644:1, L0455:1,L0370:1, T0042:1, H0560:1, H0538:1, L3904:1, L0804:1, L0805:1, L0653:1,L0776:1, L0659:1, L0787:1, L2264:1, H0547:1, H0648:1, H0539:1, L0745:1,S0436:1 and S0242:1. 104 HMSHC86 840402 114 S0002:4 and H0695:1. 105HMUAN45 833072 115 AR236:442, AR228:429, AR211:336, AR230:331,AR287:325, AR191:311, AR239:297, AR174:289, AR233:252, AR232:252,AR190:238, AR288:237, AR176:228, AR203:222, AR262:218, AR260:210,AR199:209, AR181:193, AR173:191, AR163:186, AR178:181, AR200:175,AR162:165, AR189:164, AR297:164, AR166:154, AR161:154, AR164:149,AR188:148, AR227:147, AR234:145, AR261:144, AR311:142, AR257:141,AR210:140, AR179:139, AR165:137, AR272:136, AR295:134, AR226:134,AR255:130, AR231:129, AR180:129, AR285:127, AR196:127, AR308:126,AR275:123, AR286:123, AR177:118, AR238:117, AR258:116, AR235:114,AR237:104, AR294:100, AR175:99, AR264:98, AR182:96, AR212:96, AR293:93,AR185:92, AR291:90, AR267:79, AR229:78, AR061:75, AR240:74, AR060:74,AR256:73, AR269:65, AR104:64, AR247:63, AR274:60, AR201:60, AR300:59,AR033:56, AR263:53, AR289:51, AR183:49, AR193:48, AR290:43, AR195:41,AR316:40, AR270:40, AR296:37, AR282:36, AR312:34, AR197:33, AR218:33,AR277:29, AR299:29, AR055:28, AR089:28, AR250;27, AR268:27, AR207:26,AR309:25, AR053:25, AR252:24, AR266:21, AR213:20, AR242:19, AR224:19,AR219:19, AR313:18, AR223:18, AR169:18, AR222:17, AR171:17, AR168:16,AR172:16, AR205:15, AR217:14, AR096:14, AR245:14, AR214:14, AR204:12,AR225:12, AR170:11, AR246:11, AR254:11, AR198:11, AR283:10, AR192:10,AR216:10, AR271:9, AR253:9, AR215:8, AR221:7, AR039:5, AR243:4, AR184:1,AR310:1 H0271:5, H0083:3, L0794:3, H0656:2, H0457:2, H0179:2, L0791:2,H0521:2, L0744:2, H0707:2, H0265:1, H0556:1, H0657:1, H0449:1, H0580:1,S0046:1, H0411:1, H0437:1, H0333:1, H0486:1, H0250:1, S6028:1, H0615:1,H0628:1, L0055:1, H0040:1, H0634:1, S0144:1, H0529:1, L0769:1, L0768:1,L0766:1, L0803:1, L0653:1, L0793:1, L0666:1, S0052:1, H0689:1, H0522:1,H0436:1, L0743:1, L0749:1, L0779:1, H0445:1 and H0542:1. 106 HMVBC31825598 116 AR055:5, AR316:4, AR218:3, AR282:3, AR104:2, AR283:2,AR185:2, AR096:2, AR313:2, AR240:2, AR060:2, AR300:2, AR299:1, AR277:1,AR089:1, AR039:1 L0748:10, H0556:5, S0442:5, L0438:4, L0439:4, L0754:4,H0050:3, H0040:3, L0769:3, L0806:3, L0757:3, L0759:3, L0601:3, T0002:2,S0418:2, S0358:2, S0360:2, H0580:2, S0476:2, H0549:2, H0644:2, H0529:2,L0773:2, L0768:2, L0766:2, L0805:2, L0776:2, L0663:2, L0740:2, L0747:2,L0749:2, S0436:2, H0717:1, S0212:1, H0484:1, H0661:1, S0376:1, H0729:1,H0733:1, S0007:1, H0643:1, L0622:1, L3653:1, H0013:1, H0042:1, H0052:1,L0157:1, L0471:1, H0373:1, H0083:1, H0266:1, T0006:1, H0090:1, H0268:1,H0494:1, H0509:1, H0633:1, H0646:1, S0422:1, S0002:1, L0761:1, L0772:1,L0643:1, L0644:1, L0794:1, L0803:1, L0555:1, L0659:1, L0783:1, L0809:1,L5622:1, H0690:1, H0658:1, S0328:1, S0330:1, S0152:1, H0521:1, H0696:1,S0044:1, S0027:1, L0780:1, L0752:1, L0753:1, L0755:1, S0434:1, L0485:1,H0667:1, S0276:1 and S0456:1. 107 HMWBL03 822861 117 AR313:27, AR299:16,AR219:16, AR218:14, AR316:12, AR300:6, AR039:5, AR089:5, AR055:5,AR060:4, AR240:4, AR096:4, AR282:4, AR283:3, AR277:3, AR185:3, AR104:2S0422:18, L0766:7, H0341:5, S0356:5, H0543:5, H0591:4, H0656:3, S0354:3,H0013:3, T0042:3, H0659:3, L0748:3, L0750:3, L0777:3, S0418:2, S0442:2,S0444:2, S0410:2, L0471:2, H0040:2, H0063:2, H0494:2, L0646:2, L0626:2,L0806:2, L0655:2, L0663:2, S0374:2, H0547:2, S0206:2, L0756:2, S0436:2,L0588:2, H0624:1, H0171:1, S0342:1, H0650:1, S0360:1, T0008:1, H0733:1,S0046:1, H0257:1, H0263:1, L0735:1, H0046:1, L0157:1, H0039:1, H0068:1,H0135:1, H0090:1, T0041:1, H0560:1, S0440:1, H0529:1, L0640:1, L0771:1,L0768:1, L0634:1, L0529:1, L5623:1, L0666:1, L0665:1, H0520:1, H0519:1,S0328:1, S0152:1, S0406:1, L0751:1, L0747:1, L0759:1, L0591:1, L0608:1,H0542:1, H0423:1 and H0721:1. 108 HNFGR08 825417 118 AR055:5, AR060:4,AR185:3, AR240:3, AR300:2, AR104:2, AR282:2, AR089:2, AR283:2, AR219:2,AR218:2, AR316:1, AR039:1, AR096:1 H0271:1 109 HNGAK51 603910 119AR313:60, AR039:47, AR299:29, AR277:29, AR089:26, AR185:24, AR096:23,AR240:20, AR300:19, AR316:17, AR218:14, AR060:14, AR219:14, AR104:14,AR055:11, AR282:10, AR283:6 S0052:1 110 HNGDX18 1145071 120 AR228:8,AR176:7, AR161:6, AR162:6, AR163:6, AR251:5, AR223:5, AR151:5, AR171:5,AR225:4, AR060:4, AR267:4, AR055:4, AR216:4, AR261:4, AR235:4, AR236:4,AR268:4, AR230:4, AR269:4, AR288:4, AR191:4, AR052:4, AR182:4, AR221:4,AR239:4, AR254:3, AR242:3, AR255:3, AR312:3, AR233:3, AR287:3, AR272:3,AR262:3, AR165:3, AR271:3, AR244:3, AR178:3, AR229:3, AR164:3, AR173:3,AR257:3, AR290:3, AR274:3, AR266:3, AR061:3, AR297:3, AR166:3, AR282:3,AR198:3, AR053:3, AR231:3, AR199:3, AR291:3, AR177:3, AR201:3, AR214:3,AR264:3, AR247:3, AR196:3, AR224:3, AR190:3, AR174:3, AR270:3, AR296:2,AR286:2, AR300:2, AR309:2, AR203:2, AR089:2, AR200:2, AR294:2, AR289:2,AR249:2, AR311:2, AR240:2, AR168:2, AR293:2, AR238:2, AR188:2, AR217:2,AR175:2, AR285:2, AR179:2, AR234:2, AR310:2, AR185:2, AR033:2, AR298:2,AR260:2, AR226:2, AR316:2, AR227:2, AR222:2, AR313:2, AR265:2, AR197:2,AR277:2, AR237:2, AR189:2, AR295:2, AR299:2, AR193:2, AR283:2, AR172:2,AR183:2, AR275:2, AR232:2, AR211:2, AR253:2, AR210:2, AR104:2, AR096:2,AR213:1, AR258:1, AR292:1, AR308:1, AR273:1, AR194:1, AR180:1, AR184:1,AR284:1, AR252:1, AR205:1 H0457:4, S0052:4, H0271:3, L0766:3, H0543:3,H0255:2, H0402:2, H0253:2, L0805:2, L0754:2, H0422:2, H0583:1, H0650:1,H0656:1, H0484:1, H0483:1, H0254:1, L3659:1, S0442:1, S0360:1, H0580:1,S0140:1, H0747:1, H0393:1, H0486:1, H0250:1, H0618:1, H0050:1, H0630:1,H0719:1, H0182:1, H0063:1, H0087:1, H0264:1, H0488:1, H0487:1, L0351:1,50041:1, S0448:1, S0002:1, L0761:1, L0378:1, L0655:1, L4501:1, H0539:1,S0188:1, S0146:1, H0707:1, L0599:1, H0136:1, H0423:1 and H0677:1.HNGDX18 866177 192 111 HNGGP65 597449 121 AR313:17, AR039:13, AR219:10,AR299:10, AR300:10, AR185:9, AR096:9, AR104:8, AR089:8, AR218:8,AR060:8, AR055:7, AR277:7, AR282:7, AR316:6, AR240:6, AR283:4 S0052:1112 HNGIV64 561572 122 AR185:8, AR039:8, AR060:8, AR313:7, AR055:7,AR096:6, AR300:6, AR089:6, AR240:6, AR218:6, AR299:6, AR277:6, AR316:5,AR104:5, AR283:4, AR282:3, AR219:1 S0052:1 113 HNGMW45 838613 123AR316:3 S0428:1 114 HNGPJ25 834942 124 AR060:7, AR055:7, AR218:6,AR240:6, AR282:5, AR185:5, AR277:5, AR300:5, AR299:4, AR283:3, AR089:3,AR104:3, AR316:3, AR096:3, AR039:2, AR313:2, AR219:2 H0251:8, H0624:4,L0752:4, H0286:1, L0598:1, S0428:1 and H0144:1. 115 HNHEN82 836157 125AR055:5, AR060:4, AR300:4, AR104:3, AR282:2, AR283:2, AR219:2, AR089:2,AR039:2, AR185:2, AR299:2, AR218:2, AR313:2, AR316:2, AR277:1, AR240:1116 HNHFE71 834487 126 AR055:9, AR060:8, AR218:6, AR300:5, AR185:5,AR240:5, AR277:5, AR299:5, AR282:5, AR104:5, AR283:4, AR089:4, AR039:4,AR316:4, AR096:3, AR313:3, AR219:2 S0053:1 117 HNHKV56 800877 127S0216:1 and L0746:1. 118 HOACG07 792928 128 AR202:138, AR194:120,AR315:99, AR281:99, AR198:98, AR244:88, AR246:86, AR243:85, AR205:85,AR192:82, AR241:80, AR280:79, AR204:73, AR265:70, AR283:66, AR206:63,AR310:59, AR263:57, AR271:57, AR314:56, AR273:54, AR053:50, AR052:49,AR282:49, AR033:48, AR277:47, AR275:46, AR213:45, AR274:44, AR312:43,AR316:43, AR309:42, AR104:40, AR240:40, AR300:40, AR289:39, AR096:39,AR266:38, AR251:37, AR247:36, AR089:36, AR299:36, AR284:35, AR313:35,AR186:34, AR039:33, AR055:33, AR175:32. AR219:31, AR295:31, AR291:29,AR177:28, AR218:28, AR185:27, AR268:26, AR285:26, AR270:25, AR286:25,AR253:24, AR183:24, AR060:24, AR232:23, AR061:23, AR292:23, AR298:23,AR258:22, AR256:20, AR248:20, AR296:19, AR182:19, AR238:18, AR269:18,AR290:18, AR294:18, AR267:17, AR231:17, AR226:17, AR259:17, AR229:16,AR227:16, AR234:16, AR293:15, AR233:15, AR237:14, AR249:13, AR184:13,AR179:11 L0748:4, L0749:4, H0265:3, S0442:2, H0587:2, H0427:2, S0142:2,L0769:2, L0761:2, L0800:2, L0794:2, L0657:2, L0659:2, L0663:2, L0665:2,H0689:2, L0731:2, H0677:2, H0713:1, H0716:1, H0657:1, H0661:1, H0638:1,S0360:1, H0722:1, H0122:1, H0545:1, H0009:1, H0123:1, S0388:1, H0252:1,T0023:1, T0006:1, H0135:1, H0163:1, H0412:1, L0351:1, S0440:1, H0529:1,L0372:1, L0646:1, L0645:1, L0764:1, L0662:1, L0767:1, L0766:1, L0649:1,L0803:1, L0776:1, L0655:1, L0382:1, L0809:1, L0789:1, H0672:1, H0627:1,L0751:1, L0750:1, L0753:1, L0757:1, L0758:1 and L0759:1. 119 HODBB70520196 129 AR055:7, AR218:6, AR060:5, AR104:5, AR240:4, AR300:4,AR299:4, AR096:4, AR219:4, AR283:4, AR039:3, AR185:3, AR089:3, AR316:3,AR282:3, AR277:2 H0328:1, L0789:1, L0742:1 and L0439:1. 120 HOEBK60789396 130 AR219:21, AR215:19, AR316:12, AR313:10, AR039:9, AR096:8,AR089:8, AR299:7, AR240:7, AR055:7, AR300:7, AR060:7, AR282:6, AR185:5,AR283:5, AR104:4, AR277:4 L0439:10, L0731:7, L0605:6, L0766:5, L0803:5,L0471:4, L0655:4, L0659:4, L0756:4, S0408:3, S0440:3, H0648:3, L0777:3,H0170:2, S0420:2, L0021:2, H0014:2, T0010:2, L0143:2, H0090:2, H0591:2,H0641:2, L0638:2, L0662:2, L0794:2, L0776:2, L0657:2, L0809:2, L0666:2,L0663:2, H0547:2, S0126:2, H0518:2, L0751:2, L0755:2, L0758:2, L0588:2,H0543:2, H0624:1, H0740:1, S0430:1, H0657:1, H0656:1, S0212:1, S0110:1,H0661:1, H0663:1, S0442:1, S0358:1, S0376:1, S0360:1, S0476:1, L0717:1,L0586:1, T0114:1, H0427:1, L0105:1, H0318:1, S0474:1, H0581:1, H0052:1,H0309:1, L0157:1, H0024:1, H0071:1, H0275:1, H0354:1, S6028:1, H0266:1,S0003:1, H0328:1, H0615:1, H0428:1, H0031:1, H0169:1, H0163:1, H0038:1,H0040:1, H0551:1, H0272:1, T0041:1, S0014:1, S0438:1, H0646:1, S0142:1,S0210:1, S0426:1, H0529:1, L0372:1, L0645:1, L0764:1, L0651:1, L0656:1,L5623:1, L0789:1, L0665:1, H0520:1, H0689:1, H0682:1, H0435:1, H0659:1,S0380:1, H0696:1, L0740:1, L0754:1, L0746:1, L0750:1, L0779:1, L0752:1,S0260:1, S0434:1, S0436:1, L0485:1 and H0423:1. 121 HOFAA78 836646 131AR316:60 122 HOFNB74 762821 132 AR277:18, AR283:18, AR282:14, AR313:12,AR316:11, AR055:9, AR089:9, AR240:8, AR104:8, AR218:8, AR299:8, AR096:8,AR219:8, AR300:7, AR185:7, AR039:6, AR060:5 H0415:1 123 HORBS82 638293133 H0706:2, L0809:2, S0360:1, L0623:1, H0122:1, H0041:1, H0095:1,H0292:1, H0424:1, S0364:1, L0794:1, L0787:1, L0663:1, H0780:1, H0435:1,L0743:1, L0747:1 and L0731:1. 124 HOSDO75 862049 134 AR060:6, AR055:6,AR218:6, AR240:5, AR277:5, AR300:4, AR185:4, AR299:4, AR283:4, AR089:4,AR282:3, AR104:3, AR316:3, AR096:3, AR313:2, AR039:2, AR219:2 L0766:2,L0362:2, 50358:1, H0580:1, S0046:1, H0266:1, S0003:1, H0553:1 S0344:1,L0761:1, L0794:1, S0152:1, L0777:1 and L0755:1. 125 HOUDR07 745404 135AR177:34, AR197:30, AR195:28, AR207:25, AR171:23, AR182:23, AR192:23,AR275:22, AR199:22, AR169:21, AR170:21, AR226:21, AR183:20, AR189:19,AR168:18, AR263:18, AR179:18, AR174:18, AR175:18, AR176:18, AR224:18,AR269:17, AR235:17, AR311:17, AR270:16, AR231:16, AR214:16, AR222:16,AR181:16, AR225:16, AR229:16, AR190:15, AR196:15, AR271:15, AR237:15,AR309:15, AR261:15, AR061:15, AR295:15, AR245:15, AR210:14, AR201:14,AR230:14, AR173:14, AR221:13, AR191:13, AR283:13, AR161:13, AR223:13,AR291:13, AR165:13, AR308:13, AR213:13, AR268:13, AR162:13, AR164:13,AR266:13, AR166:13, AR240:13, AR163:13, AR195:13, AR277:13, AR217:12,AR264:12, AR289:12, AR247:12, AR211:12, AR239:12, AR236:12, AR212:12,AR216:12, AR288:12, AR172:12, AR286:12, AR205:11, AR178:11, AR218:11,AR228:11, AR215:11, AR312:11, AR238:11, AR243:11, AR258:11, AR246:11,AR316:11, AR297:11, AR287:10, AR053:10, AR285:10, AR227:10, AR204:10,AR193:10, AR282:10, AR180:10, AR242:9, AR089:9, AR039:9, AR267:9,AR296:9, AR290:9, AR234:9, AR188:9, AR272:9, AR033:9, AR233:8, AR299:8,AR293:8, AR300:8, AR255:8, AR262:8, AR254:8, AR256:7, AR200:7, AR203:7,AR252:7, AR253:7, AR219:7, AR313:7, AR257:7, AR274:7, AR260:6, AR104:6,AR055:6, AR250:6, AR096:6, AR294:6, AR060:6, AR185:6, AR232:6 S0212:10,L0659:10, L0755:6, L0731:6, H0599:5, L0757:5, L0775:4, L0603:4, H0713:3,S0132:3, H0427:3, S0312:3, H0622:3, H0553:3, H0628:3, L5565:3, L0439:3,L0740:3, S0040:2, H0717:2, H0587:2, H0635:2, T0010:2, H0266:2, S0314:2,H0163:2, L0770:2, L3904:2, L0804:2, L5622:2, L0751:2, L0747:2, L0750:2,H0668:2, S0194:2, H0716:1, S0116:1, H0661:1, S0358:1, S0360:1, H0730:1,H0728:1, S0045:1, S0046:1, S0476:1, H0456:1, H0549:1, H0431:1, H0370:1,H0592:1, H0632:1, L0623:1, H0486:1, L0021:1, H0706:1, H0590:1, H0618:1,H0705:1, T0071:1, S0049:1, H0545:1, H0086:1, H0024:1, N0007:1, L3647:1,H0510:1, H0594:1, H0124:1, H0551:1, S0352:1, S0438:1, H0509:1, H0647:1,L0769:1, L0637:1, L0764:1, L0773:1, L0378:1, L0661:1, L4558:1, L0783:1,L0788:1, H0144:1, H0519:1, H0555:1, S0390:1, S0028:1, L0753:1, L0758:1,S0434:1 and S0276:1. 126 HPEAD23 773409 136 AR277:68, AR283:65,AR219:61, AR316:49, AR218:41, AR059:41, AR104:39, AR299:38, AR055:36,AR185:36, AR039:34, AR282:33, AR240:31, AR096:31, AR313:31, AR060:30,AR300:25 H0585:18, L0779:3, L0775:2, S0374:2, H0341:1, S0358:1, S0360:1,S0408:1, H0559:1, T0039:1, H0156:1, H0253:1, S0182:1, H0318:1, H0545:1,H0083:1, H0165:1, L0768:1, L0774:1, L0750:1, L0752:1 and S0031:1. 127HPFCI36 855966 137 AR218:18, AR219:16, AR313:14, AR089:9, AR055:7,AR282:6, AR060:6, AR316:6, AR185:6, AR299:5, AR240:5, AR039:5, AR300:4,AR104:4, AR283:4, AR096:4, AR277:2 L0591:4, L0754:3, H0450:2, H0486:2,H0046:2, S0003:2, H0494:2, S0422:2, L0659:2, S0126:2, H0659:2, L0750:2,L0601:2, H0170:1, H0556:1, H0657:1, S0420:1, S0354:1, H0734:1, H0749:1,H0455:1, H0403:1, H0600:1, H0013:1, H0156:1, H0599:1, H0744:1, H0082:1,S0214:1, H0622:1, H0031:1, H0673:1, H0169:1, H0090:1, H0038:1, H0022:1,H0560:1, L0643:1, L0771:1, L0773:1, L0655:1, L0807:1, L3872:1, L0792:1,L0665:1, L3811:1, S0378:1, H0518:1, S0152:1, H0521:1, L0748:1, L0749:1,L0757:1, L0759:1, S0434:1, L0596:1, L0605:1 and H0653:1. 128 HPFDI37862056 138 AR055:5, AR218:5, AR060:4, AR299:3, AR300:3, AR283:3,AR104:3, AR282:3, AR240:3, AR039:2, AR219:2, AR277:2, AR185:2, AR316:2,AR089:2, AR313:2, AR096:1 L0771:13, L0752:12, L0748:9, L0731:7, S0360:6,L0769:6, S0358:5, H0318:5, L0770:5, L0747:5, L0758:5, L0599:5, H0140:4,H0545:4, H0673:4, L0774:4, L0655:4, L0659:4, L0664:4, L0665:4, H0659:4,H0648:4, L0740:4, L0754:4, L0588:4, H0662:3, H0169:3, H0413:3, L0638:3,L0775:3, L0783:3, L0666:3, L0663:3, H0660:3, H0521:3, L0749:3, L0750:3,L0757:3, H0543:3, H0170:2, S0110:2, H0574:2, H0581:2, H0535:2, L0065:2,H0647:2, S0344:2, L0763:2, L0764:2, L0662:2, L0767:2, L0803:2, L0806:2,L0776:2, S0374:2, S0328:2, S0380:2, H0576:2, S0028:2, L0591:2, H0352:2,H0265:1, T0002:1, H0686:1, H0685:1, H0295:1, H0657:1, L0760:1, S0212:1,H0484:1, H0177:1, H0638:1, L0617:1, L0005:1, S0442:1, S0376:1, H0637:1,H0411:1, H0370:1, H0587:1, H0632:1, T0109:1, H0156:1, H0085:1, H0327:1,H0530:1, H0046:1, H0041:1, S0388:1, H0630:1, H0271:1, H0644:1, H0628:1,H0181:1, H0617:1, H0674:1, H0068:1, H0040:1, H0488:1, L0564:1, T0041:1,H0494:1, H0633:1, S0144:1, S0210:1, S0422:1, L0369:1, L0762:1, L0372:1,L0646:1, L0765:1, L0363:1, L0768:1, L0651:1, L0653:1, L0629:1, L0657:1,L0526:1, L0532:1, S0053:1, S0216:1, H0144:1, H0519:1, S0126:1. H0689:1,H0690:1, H0684:1, S0027:1, L0742:1. L0756:1, L0780:1, L0755:1, H0444:1,H0445:1, L0596:1, L0605:1, L0592:1, L0593:1, L0362:1, L0603:1 andH0136:1. 129 HPIAA80 829972 139 AR218:13, AR219:11, AR282:9, AR089:8,AR055:8, AR240:7, AR104:6, AR060:6, AR283:6, AR277:6, AR039:6, AR316:5,AR299:5, AR096:4, AR185:4, AR300:4, AR313:3 L0750:3, H0672:2, L0744:2,H0587:1, L0021:1, S0010:1, H0024:1, H0266:1, S0364:1, H0068:1, H0038:1,T0004:1, H0625:1, S0150:1, L0769:1, L0667:1, L0649:1, L0784:1, L0526:1,L0790:1, L0792:1, L0793:1, L0663:1, H0696:1, L0747:1, L0608:1 andS0276:1. 130 HPJCW58 612866 140 AR055:8, AR060:6, AR282:6, AR218:5,AR104:4, AR300:4, AR240:4. AR283:4, AR219:4, AR299:3, AR089:3, AR185:3,AR316:3, AR096:3, AR039:2, AR277:2, AR313:1 S0152:1 131 HPRBH85 695752141 AR284:14, AR295:10, AR271:8, AR293:7, AR246:7, AR243:7, AR291:7,AR244:6, AR286:6, AR290:6, AR241:6, AR285:6, AR206:6, AR310:5, AR249:5,AR273:5, AR198:5, AR280:5, AR312:5, AR186:5. AR270:5, AR294:5, AR204:5,AR269:5, AR251:5, AR202:5, AR292:5, AR275:4, AR033:4, AR253:4, AR053:4,AR182:4, AR314:4, AR259:4, AR315:4, AR298:4, AR265:4, AR309:4, AR282:4,AR274:4, AR183:4, AR061:4, AR205:4, AR296:4, AR289:4, AR052:4, AR313:4,AR175:3, AR213:3, AR238:3, AR268:3, AR248:3, AR055:3, AR177:3, AR247:3,AR231:3, AR104:3, AR256:3, AR226:2, AR185:2, AR283:2, AR277:2, AR267:2,AR227:2, AR300:2, AR096:2, AR089:2, AR258:2, AR219:2, AR263:2, AR299:2,AR237:2, AR240:2, AR060:2, AR316:2, AR218:2, AR281:2, AR039:2, AR233:1,AR232:1, AR184:1, AR234:1, AR266:1, AR192:1 L0439:5, L0740:4, L0777:4,L0755:4, L0794:2, L0803:2, L0438:2, L0602:2, L0752:2, L0599:2, H0713:1,H0583:1, 50360:1, L3653:1, L0471:1, H0510:1, H0032:1, H0488:1, H0413:1,L0662:1, L0804:1, L0775:1, L0805:1, L0655:1, L0809:1, L0519:1, S0148:1,H0547:1, L0747:1, L0686:1 and H0665:1. 132 HPRCA64 824074 142 AR295:13,AR285:10, AR297:8, AR191:8, AR291:7, AR261:7, AR288:7, AR189:6, AR296:6,AR188:6, AR178:6, AR271:6, AR170:5, AR181:5, AR165:5, AR175:5, AR174:5,AR270:5, AR235:5, AR223:5, AR164:5, AR190:5, AR166:5, AR272:5, AR172:5,AR210:5, AR255:5, AR236:5, AR274:5, AR196:5, AR217:5, AR287:5, AR246:4,AR096:4, AR089:4, AR286:4, AR176:4, AR168:4, AR195:4, AR269:4, AR183:4,AR173:4, AR060:4, AR219:4, AR222:4, AR214:3, AR199:3, AR268:3, AR033:3,AR218:3, AR257:3, AR104:3, AR161:3, AR163:3, AR275:3, AR171:3, AR240:3,AR299:3, AR182:3, AR289:3, AR290:3, AR177:3, AR316:3, AR207:3, AR245:3,AR180:3, AR162:3, AR300:3, AR216:3, AR262:3, AR293:3, AR197:3, AR282:3,AR258:3, AR179:3, AR294:3, AR200:2, AR185:2, AR238:2, AR201:2, AR203:2,AR225:2, AR267:2, AR193:2, AR232:2, AR260:2, AR266:2, AR231:2, AR211:2,AR205:2, AR283:2, AR053:2, AR264:2, AR256:2, AR247:2, AR192:2, AR237:2,AR226:2, AR243:2, AR204:1, AR277:1, AR239:1, AR229:1, AR169:1, AR313:1L0005:7, L0662:7, L0665:7, L0666:6, L0740:6, S0222:5, L0659:5, L0439:5,L0483:4, H0547:4, L0754:4, L0756:4, L0779:4, S0194:4, S0049:3, L0646:3,L0521:3, L0663:3, L0664:3, L0438:3, H0696:3, L0777:3, H0171:2, S0356:2,S0442:2, S0360:2, S0408:2, H0052:2, H0563:2, L0471:2, S0388:2, S0051:2,S6028:2, H0266:2, H0623:2, S0440:2, L0598:2, L0520:2, L0641:2, L0771:2,L0768:2, L0774:2, L0805:2, L0776:2, L0518:2, H0670:2, H0648:2, H0672:2,S0028:2, L0751:2, L0731:2, L0758:2, S0031:2, L0596:2, S0026:2, S0196:2,H0624:1, H0170:1, H0686:1, H0685:1, H0717:1, H0381:1, S0212:1, H0662:1,S0354:1, S0376:1, S0045:1, S0046:1, H0411:1, H0369:1, H0602:1, T0040:1,H0013:1, H0427:1, H0390:1, S0474:1, H0545:1, H0046:1, H0178:1, H0562:1,H0123:1, H0201:1, S0003:1, H0428:1, T0006:1, H0031:1, H0553:1, H0032:1,H0163:1, H0551:1, L0564:1, S0370:1, S0450:1, L0769:1, L0637:1, L5565:1,L0372:1, L0773:1, L0650:1, L0806:1, L0527:1, L0526:1, L0783:1, L0809:1,S0374:1, L0565:1, H0519:1, H0682:1, H0435:1, H0659:1, H0660:1, S0328:1,S0330:1, S0380:1, L0602:1, H0555:1, L0753:1, L0755:1, L0759:1, S0260:1,S0434:1, S0436:1, L0595:1, H0667:1 and S0242:1. 133 HPRCD35 853551 143AR104:14, AR089:11, AR055:11, AR185:10, AR219:10, AR299:10, AR218:10,AR313:10, AR282:10, AR240:9, AR316:9, AR283:9, AR096:8, AR060:7,AR039:6, AR300:5, AR277:5 L0748:5, L0754:5, L0803:4, L0805:4, L0777:4,L0662:3, L0766:3, H0556:2, H0013:2, H0551:2, H0264:2, L0800:2, L0806:2,L0664:2, L0439:2, L0756:2, L0758:2, S0192:2, H0657:1, S0442:1, S0358:1,S0045:1, S0046:1, H0747:1, H0550:1, H0392:1, S0280:1, H0575:1, H0545:1,H0046:1, H0050:1, H0644:1, H0617:1, L0055:1, H0032:1, H0212:1, H0038:1,H0560:1, S0438:1, S0210:1, L0769:1, L0796:1, L5575:1, L0768:1, L0794:1,L0649:1, L0776:1, L0659:1, L0542:1, L0526:1, L0663:1, H0144:1, L0565:1,L3811:1, H0683:1, H0659:1, S0152:1, H0521:1, H0522:1, H0540:1, S0118:1,S0032:1, L073L1, L07591 and H0668:1. 134 HPTRM02 812879 144 H0617:7,H0087:6, H0657:5, S0410:3, L0754:3, S0356:2, L0717:2, H0150:2, H0687:2,H0424:2, H0551:2, L0769:2, L0774:2, L0743:2, L0758:2, L0592:2, H0556:1,T0002:1, H0686:1, H0685:1, T0049:1, H0663:1, S0442:1, S0444:1, S0360:1,S0476:1, H0550:1, H0486:1, H0250:1, L0021:1, T0048:1, S0474:1, S0049:1,H0052:1, H0309:1, H0597:1, H0544:1, H0014:1, H0107:1, S6028:1, H0622:1,H0644:1, H0102:1, S0038:1, L0351:1, S0450:1, S0344:1, S0002:1, L0764:1,L0766:1, L0805:1, L0776:1, L0655:1, L0661:1, L0657:1, L0809:1, L0666:1,L0665:1, L2652:1, L2260:1, L2261:1, H0689:1, H0435:1, H0521:1, H0696:1,H0555:1, L0744:1, L0439:1, L0749:1, L0777:1, L0755:1, L0759:1, S0436:1,L0597:1, L0599:1, L0366:1 and S0196:1. 135 HRAAD30 866187 145 AR282:5,AR277:5, AR060:5, AR055:4, AR185:4, AR300:4, AR299:3, AR104:3, AR218:3,AR283:3, AR316:3, AR089:3, AR039:2, AR096:2, AR313:2, AR240:1 L0731:6,L2800:4, H0617:4, H0547:4, L0758:4, S0420:3, H0013:3, L0748:3, L0747:3,S0358:2, L3278:2, L0770:2, S0126:2, L0439:2, L0751:2, L0777:2, L0757:2,H0543:2, S0040:1, L3012:1, H0341:1, S0046:1, H0550:1, H0497:1, H0333:1,H0427:1, H0618:1, H0253:1, S0474:1, H0052:1, H0546:1, H0571:1, L0471:1,H0024:1, H0051:1, S6028:1, H0286:1, H0622:1, H0644:1, L0455:1, T0067:1,H0561:1, S0440:1, H0130:1, H0529:1, L0763:1, L0769:1, L0772:1, L0372:1,L0662:1, L0806:1, L0807:1, L0659:1, L5622:1, L4501:1, L0666:1, L0664:1,L0709:1, L2261:1, H0144:1, L2402:1, S0374:1, H0520:1, L3831:1, H0555:1,S0027:1, L0740:1, L0750:1, L0752:1, L0759:1, S0436:1, L0592:1, L0604:1,H0542:1, S0398:1, L3837:1 and H0677:1. 136 HRADF49 866481 146 AR244:12,AR296:6, AR205:6, AR183:6, AR292:6, AR104:5, AR249:5, AR291:5, AR285:5,AR298:5, AR206:5, AR289:4, AR240:4, AR293:4, AR275:4, AR270:4, AR295:4,AR294:4, AR284:3, AR213:3, AR186:3, AR060:3, AR286:3, AR234:3, AR229:3,AR282:3, AR267:3, AR184:3, AR096:3, AR283:3, AR033:3, AR251:3, AR300:2,AR313:2, AR316:2, AR185:2, AR039:2, AR299:2, AR218:2, AR256:2, AR089:2,AR219:2, AR061:2, AR243:2, AR055:2, AR269:2, AR277:2, AR233:2, AR238:2,AR182:2, AR268:2, AR175:2, AR259:2, AR266:2, AR232:2, AR258:2, AR227:2,AR315:2, AR263:1, AR226:1, AR309:1, AR314:1, AR053:1, AR290:1, AR052:1,AR231:1 H0618:9, L0751:7, L0754:6, L0758:6, H0253:5, L0748:5, L0439:5,H0580:3, L3816:3, H0052:3, L0770:3, L0663:3, H0556:2, H0733:2, H0351:2,H0706:2, H0567:2, H0625:2, S0142:2, L0639:2, L3905:2, L0659:2, L0543:2,L5623:2, L0749:2, S0436:2, H0423:2, L3643:1, H0381:1, S0212:1, H0254:1,H0663:1, H0638:1, S0418:1, H0741:1, H0735:1, S0045:1, S0046:1, S0476:1,S6022:1, H0549:1, H0550:1, S0222:1, H0370:1, H0497:1, H0574:1, L0622:1,L0623:1, L3655:1, H0101:1, H0427:1, S0280:1, H0122:1, H0194:1, H0596:1,H0570:1, H0081:1, H0620:1, H0014:1, H0083:1, H0355:1, H0510:1, H0424:1,H0030:1, H0553:1, H0628:1, S0364:1, S0366:1, H0038:1, H0551:1, H0100:1,L0351:1, H0494:1, S0438:1, H0633:1, S0144:1, S0422:1, L0371:1, L0769:1,L3904:1, L0772:1, L0648:1, L0497:1, L0375:1, L0511:1, L0666:1, L0709:1,L0710:1, H0144:1, L3811:1, L3824:1, H0520:1, H0593:1, H0682:1, H0670:1,H0672:1, H0539:1, L3833:1, S0044:1, H0626:1, H0732:1, S3012:1, S3014:1,S0027:1, S0028:1, L0779:1, L0584:1, L0608:1, L0593:1, H0667:1 andH0542:1. 137 HRDDQ39 840405 147 AR313:36, AR039:33, AR185:27, AR299:20,AR089:18, AR300:17, AR096:17, AR240:16, AR218:15, AR277:14, AR316:13,AR060:11, AR219:10, AR104:9, AR055:8, AR282:7, AR283:7 S0001:2, H0436:2,S0134:1, H0657:1, H0441:1, H0009:1, H0123:1, H0050:1, H0428:1, H0124:1,H0529:1, H0521:1 and H0352:1. 138 HRDEX93 816046 148 AR104:30, AR218:25,AR219:24, AR240:22, AR096:21, AR185:17, AR039:16, AR316:16, AR313:16,AR055:14, AR060:14, AR299:13, AR089:13, AR282:9, AR277:9, AR300:9,AR283:6 H0694:12, L0748:10, L0731:7, L0754:6, H0556:5, L0758:5, H0265:4,S0420:4, S0408:4, L0517:4, H0657:3, H0618:3, H0052:3, H0083:3, H0553:3,H0494:3, L0763:3, L0666:3, L0663:3, S0126:3, L0747:3, H0295:2, S0134:2,S0418:2, H0637:2, S0046:2, H0431:2, H0545:2, H0014:2, H0271:2, H0039:2,H0424:2, H0124:2, H0641:2, L0764:2, L0766:2, L0774:2, L0775:2, L0776:2,L0655:2, L0783:2, L0665:2, H0519:2, H0522:2, S0044:2, L0755:2, S0436:2,L0595:2, L0362:2, H0543:2, S0040:1, H0740:1, H0656:1, S0212:1, H0484:1,H0661:1, H0662:1, S0360:1, H0733:1, H0619:1, S0222:1, H0486:1, H0156:1,H0575:1, H0706:1, H0253:1, S0010:1, S0346:1, H0318:1, H0596:1, H0231:1,H0046:1, H0150:1, H0081:1, H0050:1, H0012:1, H0620:1, L0163:1, S0051:1,T0010:1, S6028:1, H0266:1, H0179:1, H0292:1, H0031:1, H0644:1, H0182:1,H0617:1, H0606:1, H0673:1, L0455:1, L0456:1, H0598:1, H0038:1, H0040:1,H0616:1, H0087:1, T0067:1, H0264:1, T0041:1, H0131:1, H0647:1, S0002:1,L0772:1, L0642:1, L0662:1, L0767:1, L0657:1, L0659:1, L0382:1, L5623:1,L0664:1, S0374:1, H0593:1, H0690:1, H0682:1, H0659:1, H0658:1, H0666:1,H0651:1, H0539:1, H0521:1, S0406:1, H0576:1, L0743:1, L0740:1, L0750:1,L0779:1 and H0445:1. 139 HROEA08 866190 149 S0474:57, H0521:14,L0766:12, S0422:10, L0809:8, H0069:7, H0591:7, H0556:6, H0650:5,H0749:5, H0486:5, H0090:5, L0655:5, S0114:4, S0134:4, H10747:4, S0003:4,H0268:4, H0641:4, L0770:4, H0518:4, L0777:4, H0656:3, H0638:3, H0271:3,H0039:3, L0598:3, L0763:3, L0659:3, H0436:3, L0754:3, L0756:3, L0731:3,H0423:3, H0422:3, H0265:2, H0657:2, S0354:2, H0013:2, L0105:2, H0581:2,H0622:2, H0598:2, H0100:2, S0144:2, S0002:2, H0529:2, L0648:2, L5564:2,L0803:2, L0776:2, L0789:2, L0663:2, H0696:2, H0445:2, H0542:2, H0506:2,H0624:1, H0583:1, H0305:1, H0125:1, S0360:1, H0549:1, H0586:1, L3657:1,T0060:1, H0075:1, H0002:1, H0599:1, H0004:1, H0318:1, H0421:1, H0251:1,H0457:1, H0178:1, L0471:1, T0003:1, H0024:1, S0388:1, S0051:1, S0024:1,H0266:1, H0687:1, H0644:1, H0040:1, H0264:1, T0069:1, H0625:1, S0440:1,H0646:1, S0344:1, S0426:1, L0761:1, L0641:1, L0662:1, L0794:1, L0650:1,L0774:1, L0375:1, L0784:1, L0805:1, L0515:1, H0144:1, H0702:1, S0374:1,H0520:1, S0126:1, H0689:1, H0659:1, H0658:1, S0378:1, H0S22:1, H0555:1,H0727:1, S3014:1, L0747:1, L0779:1, L0752:1, L0753:1, L0757:1, S0260:1,S0242:1, H0543:1 and S0412:1. 140 HRTAP63 780698 150 AR219:53, AR218:46,AR313:33, AR104:28, AR096:26, AR089:25, AR316:24, AR039:20, AR299:19,AR185:18, AR300:17, AR060:17, AR282:16, AR055:15, AR240:13, AR277:10,AR283:9 S0474:28, H0521:15, L0758:14, L0752:13, L0731:13, L0755:10,H0641:9, L0766:8, H0179:6, L0748:6, L0439:6, L07S9:6, H0638:5, S0222:5,H0581:5, L0662:5, L0655:5, H0436:5, L0740:S, L0777:5, S0436:5, H0457:4,L077S:4, L0809:4, H0S22:4, L0742:4, L0754:4, L0747:4, L0749:4, L0750:4,L0757:4, H0580:3, H0619:3, H0052:3, S0003:3, H0038:3, H0623:3, L0666:3,L0663:3, S0053:3, S0126:3, S0434:3, S0026:3, S0212:2, S0442:2, S0408:2,H0747:2, S0476:2, H0156:2, L0021:2, H0599:2, S0010:2, H0014:2, S0214:2,H0031:2, H0644:2, H0628:2, S0036:2, H0090:2, H0616:2, S0144:2, S0002:2,L0598:2, L0764:2, L0768:2, L0774:2, L0806:2, L0653:2, L0657:2, L0659:2,H0520:2, H0547:2, H0539:2, H0710:2, S0027:2, L0779:2, L0588:2, L0592:2,L0594:2, L0366:2, H0665:2, H0739:1, H0170:1, T0002:1, S0342:1, H0717:1,H0740:1, S0114:1, S0218:1, H0650:1, H0657:1, S0282:1, L3659:1, S0418:1,S0420:1, L0005:1, T0008:1, H0742:1, H0722:1, H0735:1, S0007:1, S0046:1,H0749:1, L3388:1, H0351:1, H0406:1, S0278:1, H0461:1, H0601:1, H0586:1,H0497:1, H0574:1, T0039:1, L3655:1, H0013:1, H0427:1, H0575:1, H0590:1,H0421:1, S0049:1, H0327:1, H0545:1, H0046:1, H0572:1, H0570:1, H0050:1,L0471:1, H0373:1, H0510:1, H0375:1, S6028:1, H0266:1, H0271:1, H0719:1,H0416:1, S0340:1, S0312:1, H0615:1, L0483:1, T0006:1, H0169:1, H0674:1,H0163:1, H0591:1, H0551:1, H0264:1, H0412:1, H0059:1, H0494:1, S0015:1,S0344:1, UNKWN:1, H0529:1, L0520:1, L0637:1, L0761:1, L0667:1, L0772:1,L0641:1, L0648:1, L0521:1, L0794:1, L0649:1, L0803:1, L0805:1, L0783:1,L0384:1, L5622:1, L0793:1, L0665:1, L0665:1, S0052:1, S0428:1, S0216:1,H0144:1, L3811:1, H0658:1, H0670:1, H0666:1, H0672:1, H0651:1, L0355:1,S0328:1, S0152:1, H0696:1, S0406:1, H0555:1, S0028:1, S0032:1, L0744:1,L0745:1, L0780:1, L0362:1, H0422:1 and H0721:1. 141 HSAVW42 637660 151AR277:28, AR283:24, AR219:20, AR055:17, AR218:16, AR316:16, AR282:16,AR313:15, AR089:15, AR104:13, AR299:12, AR185:11, AR240:11, AR096:11,AR039:10, AR300:9, AR060:8 H0412:2, S0114:1, S0222:1, H0169:1, L0520:1,L0805:1, L0776:1, L0750:1 and L0777:1. 142 HSAYC41 688057 152 S0114:1,H0411:1, H0179:1, L0665:1 and H0435:1. 143 HSLHX15 777861 153 AR060:8,AR055:7, AR218:6, AR240:5, AR300:5, AR282:4, AR185:4, AR089:4, AR283:4,AR104:4, AR299:4, AR277:3, AR316:3, AR219:3, AR096:3, AR039:2, AR3131T0040:1, L0564:1, S0028:1 and L0480:1. 144 HSNBM34 635131 154 AR185:18,AR039:18, AR299:15, AR104:11, AR055:9, AR277:9, AR060:8, AR096:8,AR282:8, AR300:7, AR218:7, AR240:7, AR313:6, AR316:6, AR219:5, AR283:4,AR089:4 H0599:9, H0144:8, H0457:7, H0266:7, H0494:6, H0046:5, H0031:5,H0553:5, L5622:5, H0593:5, H0521:5, H0734:4, H0013:4, H0135:4, S0436:4,S0212:3, H0069:3, H0036:3, H0052:3, S0022:3, H0708:3, H0551:3, H0696:3,S0434:3, H0713:2, H0717:2, S0418:2, S0354:2, H0580:2, H0728:2, H0733:2,H0550:2, H0587:2, H0559:2, H0706:2, H0253:2, H0355:2, H0039:2, S0364:2,H0038:2, H0634:2, H0433:2, H0560:2, S0440:2, H0646:2, L3818:2, S0002:2,L0506:2, L5623:2, H0547:2, S0126:2, H0518:2, H0436:2, H0478:2, S3014:2,L0601:2, H0506:2, H0265:1, H0556:1, T0002:1, S0114:1, H0583:1, S0116:1,S0356:1, S0442:1, S0358:1, S0376:1, S0360:1, S0408:1, H0340:1, H0742:1,H0735:1, S0132:1, S0476:1, H0619:1, S0278:1, S0222:1, H0409:1, H0602:1,H0592:1, H0586:1, H0486:1, H0270:1, S0280:1, H0042:1, H0575:1, H0122:1,H0590:1, S0010:1, T0048:1, H0318:1, S0474:1, S0049:1, H0173:1, H0085:1,H0597:1, H0231:1, H0327:1, H0544:1, H0123:1, H0050:1, H0095:1, H0373:1,L0163:1, H0051:1, T0010:1, T0023:1, H0213:1, L0142:1, H0383:1, S0366:1,H0163:1, H0040:1, H0616:1, H0057:1, H0379:1, S0038:1, T0042:1, S0150:1,S0144:1, H0529:1, L0369:1, L3905:1, L0766:1, L0775:1, L0532:1, H0693:1,H0689:1, H0670:1, L0602:1, H0522:1, S0044:1, L0611:1, S0027:1, S0028:1,L0741:1, L0743:1, L0748:1, L0439:1, L0592:1, L0485:1, L0608:1, L0366:1,H0668:1, H0542:1, H0543:1 and H0423:1. 145 HSSEF77 658725 155 H0617:7,L0750:7, H0556:5, L0769:5, L0783:5, L0758:5, L0759:5, L0665:4, L0741:4,S0132:3, L0761:3, L0742:3, L0439:3, L0755:3, L0592:3, H0618:2, H0620:2,H0038:2, L0771:2, L0662:2, L0659:2, L0666:2, S0126:2, H0670:2, S0328:2,S0380:2, L0747:2, L0753:2, L0731:2, H0395:1, H0295:1, H0294:1, H0657:1,H0656:1, H0341:1, H0484:1, H0663:1, H0638:1, S0356:1, S0444:1, H0741:1,L3271:1, H0549:1, H0550:1, H0370:1, H0455:1, H0632:1, H0486:1, T0039:1,T0112:1, H0156:1, H0581:1, H0052:1, H0545:1, H0046:1, H0150:1, H0081:1,S0051:1, H0107:1, H0061:1, H0188:1, H0288:1, S0250:1, H0428:1, H0135:1,L0766:1, L5574:1, L0774:1, L0775:1, L0375:1, L0806:1, L0776:1, L0807:1,L0657:1, L0658:1, L0540:1, L0384:1, L0809:1, L0663:1, L0438:1, H0672:1,H0754:1, S0188:1, S0406:1, H0436:1, H0576:1, S3014:1, L0748:1, L0779:1,L0757:1 and H0506:1. 146 HT4FV41 853400 156 AR244:10, AR185:10, AR204:9,AR275:9, AR202:9, AR194:9, AR052:9, AR271:8, AR246:8, AR289:8, AR219:7,AR316:7, AR104:7, AR198:7, AR089:7, AR277:7, AR310:7, AR060:7, AR309:7,AR206:7, AR039:7, AR229:7, AR282:7, AR053:7, AR283:7, AR269:6, AR205:6,AR270:6, AR312:6, AR184:6, AR186:6, AR299:6, AR096:6, AR240:6, AR251:6,AR274:6, AR182:6, AR055:6, AR033:6, AR291:6, AR218:6, AR243:6, AR266:6,AR290:6, AR192:5, AR268:5, AR280:5, AR247:5, AR213:5, AR298:5, AR231:5,AR273:5, AR313:5, AR234:5, AR284:5, AR296:5, AR061:5, AR248:5, AR238:5,AR285:5, AR183:5, AR253:5, AR300:5, AR267:4, AR286:4, AR315:4, AR175:4,AR293:4, AR294:4, AR177:4, AR249:4, AR281:3, AR227:3, AR233:3, AR292:3,AR263:3, AR265:3, AR232:3, AR295:3, AR226:3, AR237:3, AR258:2, AR314:2,AR256:1, AR259:1, AR179:1, AR241:1 L0794:10, L0800:7, L0769:6, L0751:6,L0761:4, L0809:4, H0521:4, L0439:4, H0585:3, H0617:3, H0494:3, L0659:3,L0665:3, L0777:3, H0265:2, H0255:2, S0354:2, S0376:2, H0370:2, H0069:2,H0083:2, H0040:2, L0770:2, L0764:2, L0662:2, L5622:2, L0666:2, L0663:2,L0438:2, L0743:2, L0780:2, S0436:2, H0667:2, H0423:2, S0040:1, H0713:1,H0295:1, H0254:1, H0638:1, S0418:1, S0420:1, S0360:1, H0734:1, S0046:1,S0476:1, H0587:1, H0635:1, H0575:1, H0004:1, H0618:1, H0052:1, H0194:1,H0544:1, H0545:1, H0373:1, T0010:1, H0267:1, H0179:1, H0622:1, L0194:1,H0181:1, H0124:1, H0087:1, H0412:1, H0413:1, H0646:1, S0144:1, L0369:1.L0640:1, L0763:1, L0772:1, L0646:1, L0643:1, L0644:1, L0768:1, L0803:1,L0805:1, L0655:1, L0518:1, L0783:1, L0384:1, L0789:1, S0052:1, L2263:1,L0710:1, H0547:1, H0682:1, S0152:1, H0187:1, H0727:1, S0390:1, L0752:1,L0757:1, L0758:1, H0665:1, H0543:1, H0422:1 and S0424:1. 147 HT5FX79794169 157 AR313:23, AR055:11, AR089:11, AR316:10, AR060:10, AR039:9,AR277:9, AR096:8, AR240:8, AR283:8, AR299:7, AR300:7, AR282:7, AR185:7,AR104:6, AR219:5, AR218:4 H0584:45, L0748:8, H0167:6, L0766:6, L0779:5,L0758:5, H0445:5, L0777:4, H0581:3, H0529:3, L0805:3, L0789:3, L0750:3,H0333:2, L0471:2, H0024:2, L0483:2, H0090:2, H0494:2, L0769:2, L0768:2,L0774:2, L0783:2, L5622:2, H0593:2, L0747:2, L0755:2, L0731:2, L0589:2,H0556:1, S0114:1, S0134:1, H0255:1, L0481:1, S0360:1, H0675:1, H0645:1,H0619:1, H0453:1, H0574:1, H0575:1, H0309:1, L0157:1, H0014:1, H0083:1,H0615:1, H0622:1, H0553:1, H0708:1, H0598:1, H0038:1, H0616:1, H0551:1,H0477:1, H0056:1, S0016:1, H0561:1, S0002:1, L0763:1, L0637:1, L0761:1,L0646:1, L0771:1, L0794:1, L0803:1, L0375:1, L0806:1, L0653:1, L0776:1,L0655:1, L0527:1, L0790:1, L0792:1, L4501:1, L4508:1, H0547:1, H0689:1,H0690:1, H0659:1, H0539:1, S0380:1, H0518:1, H0521:1, H0696:1, S0406:1,L0754:1, L0749:1, S0394:1, S0106:1, S0026:1, S0276:1, H0542:1, H0543:1,H0423:1, S0424:1 and H0506:1. 148 HT5GR59 801930 158 AR240:19, AR096:15,AR316:10, AR300:9, AR055:9, AR039:8, AR313:8, AR282:8, AR277:7, AR185:7,AR060:7, AR219:7, AR218:6, AR299:6, AR104:6, AR283:6, AR089:5 H0584:36,H0585:22, H0141:11, H0167:9, H0457:7, H0521:6, S0474:4, H0575:3,L0731:3, H0265:2, H0556:2, H0581:2, L0761:2, H0543:2, H0140:1, H0638:1,S0358:1, S0140:1, H0747:1, H0619:1, H0497:1, H0559:1, H0069:1, H0635:1,H0427:1, S0280:1, H0252:1, H0477:1, L0667:1, L0768:1, L0775:1, L0659:1,L0791:1, L0792:1, S0053:1, L0777:1, L0758:1, H0445:1 and H0506:1. 149HTAE178 637684 159 AR249:6, AR060:5, AR248:5, AR241:4, AR202:4, AR055:4,AR273:4, AR244:4, AR053:4, AR184:3, AR052:3, AR253:3, AR186:3, AR182:3,AR213:3, AR206:3, AR175:3, AR251:3, AR270:3, AR282:3, AR312:3, AR274:2,ARO61:2, AR286:2, AR300:2, AR269:2, AR243:2, AR309:2, AR298:2, AR192:2,AR246:2, AR089:2, AR185:2, AR275:2, AR316:2, AR204:2, AR299:2, AR268:2,AR233:2, AR240:2, AR283:2, AR310:2, AR238:2, AR198:2, AR294:2, AR277:2,AR033:2, AR267:2, AR247:2, AR104:2, AR292:2, AR284:2, AR295:2, AR285:1,AR096:1, AR313:1, AR226:1, AR177:1, AR290:1, AR218:1, AR259:1, AR231:1,AR281:1, AR039:1, AR291:1, AR237:1, AR293:1, AR229:1, AR205:1, AR296:1H0069:1, S0474:1 and L0766:1. 150 HTEAG62 812332 160 AR310:2, AR282:2,AR206:2, AR273:2, AR186:1, AR295:1, AR294:1, AR175:1 L0766:6, H0038:5,L0758:4, H0616:3, S0422:2, L0779:2, L0752:2, H0638:1, S0376:1, S0132:1,L3388:1, H0250:1, L0564:1, L0794:1, L0803:1, L0666:1, L0777:1, L0755:1,H0595:1, S0434:1 and H0542:1. 151 HTEDJ28 762845 161 AR219:24, AR218:21,AR089:19, AR055:18, AR313:16, AR299:15, AR096:13, AR316:13, AR104:13,AR060:11, AR185:11, AR283:10, AR039:9, AR277:9, AR282:9, AR300:8,AR240:7 L0747:9, L0439:8, L0809:6, L0766:5, L0750:5, L0758:5, L0740:4,L0752:4, L0731:4, L0662:3, H0547:3, L0779:3, L0777:3, L0757:3, H0375:2,L0646:2, L0774:2, L0783:2, H0144:2, L0759:2, S0442:1, H0333:1, T0060:1,H0327:1, H0399:1, L0483:1, H0038:1, L0564:1, S0382:1, H0538:1, H0743:1,L0763:1, L0638:1, L0765:1, L0771:1, L0649:1, L0522:1, L0775:1, L0655:1,L0659:1, L0792:1, L0663:1, L0438:1, H0648:1, L0756:1, L0753:1, L0596:1,L0590:1, L0592:1, L0608:1, H0423:1 and S0460:1. 152 HTEDS12 838621 162H0253:4, L0779:2, H0618:1, H0050:1, H0038:1, L0151:1, L0758:1 andH0445:1. 153 HTBHA56 806461 163 AR104:41, AR089:32, AR299:24, AR313:21,AR055:19, AR096:18, A2R240:18, AR060:17, AR185:17, AR316:16, AR039:15,AR282:12, AR300:11, AR219:11, AR277:10, AR218:9, AR283:8 L0754:8,L0770:5, L0794:5, L0805:5, H0553:4, L0803:4, H0615:3, L0769:3, L0439:3,L0777:3, L0752:3, H0052:2, H0038:2, L0637:2, L3905:2, L0768:2, L0659:2,L5623:2, L0666:2, L0664:2, L3828:2, H0547:2, H0593:2, H0682:2, H0539:2,H0521:2, L0744:2, L0757:2, L0604:2, L0601:2, H0624:1, H0657:1, H0656:1,S0212:1, H0254:1, H0662:1, L0005:1, L2323:1, S0045:1, H0619:1, H0550:1,H0592:1, L3655:1, H0013:1, H0036:1, H0618:1, H0620:1, H0023:1, S0051:1,H0188:1, H0028:1, H0428:1, T0006:1, H0213:1, L0455:1, H0135:1, H0616:1,H0264:1, H0413:1, T0069:1, S0038:1, H0100:1, L3180:1, L0763:1, L0638:1,L5565:1, L0761:1, L0667:1, L0804:1, L0650:1, L0375:1, L0776:1, L0807:1,L0809:1, L0519:1, L0663:1, L0665:1, L0710:1, L3811:1, L3825:1, L3827:1,H0520:1, S0126:1, H0684:1, H0435:1, H0659:1, H0648:1, S0190:1, S0404:1,H0555:1, L0741:1, L0751:1, L0747:1, L0753:1 and L0758:1. 154 HTEJD29695798 164 H0038:2 155 HTENR63 877952 165 AR277:32, AR283:29, AR218:25,AR219:22, AR282:21, AR316:21, AR089:20, AR313:19, AR104:18, AR055:17,AR299:16, AR096:16, AR240:16, AR185:15, AR300:14, AR039:14, AR060:11L0748:9, L0777:6, L0439:5, L0749:5, L0766:4, L0438:4, L0755:4, L0752:3,L0594:3, L3814:2, S0212:2, H0014:2, H0032:2, H0598:2, H0038:2, H0100:2,L0775:2, S0330:2, L0754:2, L0750:2, L0731:2, L0758:2, L0759:2, L0485:2,S0192:2, S0040:1, H0583:1, S0356:1, H0733:1, S0046:1, H0747:1, L3652:1,H0613:1, H0024:1, H0373:1, H0375:1, H0179:1, H0166:1, H0673:1, H0591:1,H0616:1, H0551:1, H0412:1, H0129:1, H0529:1, L0761:1, L0771:1, L0804:1,L0784:1, L0806:1, L0655:1, L0783:1, L0666:1, H0144:1, L3811:1, S0126:1,S0328:1, H0539:1, S0152:1, L0740:1, L0756:1, L0779:1, L0757:1, H0445:1,L0599:1 and S0026:1. 156 HTGGM44 842856 166 AR246:5, AR244:5, AR184:5,AR253:5, AR309:4, AR313:4, AR186:4, AR052:3, AR206:3, AR312:3, AR310:3,AR204:3, AR274:3, AR291:3, AR060:3, AR055:3, AR229:3, AR053:3, AR292:3,AR269:3, ARO6I:3, AR243:3, AR205:3, AR247:3, AR213:2, AR299:2, AR294:2,AR089:2, AR273:2, AR270:2, AR266:2, AR263:2, AR039:2, AR265:2, AR293:2,AR316:2, AR275:2, AR282:2, AR267:2, AR251:2, AR277:2, AR231:2, AR283:2,AR238:2, AR284:2, AR185:2, AR271:2, AR300:2, AR182:2, AR226:2, AR096:1,AR237:1, AR033:1, AR234:1, AR290:1, AR240:1, AR241:1, AR259:1, AR194:1,AR227:1, AR104:1, AR298:1 L0748:8, L0805:2, L0599:2, S0218:1, T0040:1,H0635:1, S0250:1, H0212:1, H0634:1, H0063:1, S0002:1, L0766:1, H0144:1,S0126:1 and H0518:1. 157 HTHBZ06 832477 167 AR218:86, AR219:83,AR089:54, AR104:50, AR282:48, AR313:44, AR283:40, AR055:34, AR316:31,AR240:29, AR185:27, AR096:23, AR060:22, AR299:22, AR300:16, AR039:15,AR277:11 S0414:9, L0005:7, L0065:7, S0360:6, S0422:5, H0545:4, AR0648:4,L0777:4, L0758:4, H0716:3, H0657:3, S0474:3, L0770:3, L0666:3, L0665:3,L0600:3, H0674:2, H0494:2, L0769:2, L0638:2. L0637:2, L0768:2, L0803:2,L0774:2, L0805:2, L0664:2, L0438:2, H0520:2, H0696:2. L0751:2, L0745:2,L0749:2, L0756:2, L0779:2, L0757:2, L3643:1, H0484:1, H0671:1, S0358:1,L3649:1, H0742:1, H0741:1, S0132:1, L0623:1, H0581:1, S0214:1, H0063:1,H0412:1, H0413:1, S0002:1, L0369:1, L0796:1, L0662:1, L0766:1, L0375:1,L0656:1, L0659:1, L0647:1, L3872:1, L0663:1, H0684:1, S0328:1, S0350:1,H0436:1, L0743:1, L0754:1, L0755:l, L0731:1, S0031:1, S0436:1, L0485:1,L0608:1, L0362:1, H0506:1 and H0352:1. 158 HTLBT80 840045 168 AR251:22,AR273:18, AR053:18, AR309:16, AR310:16, AR183:15, AR313:15, AR274:15,AR263:15, AR247:15, AR312:14, AR314:14, AR266:14, AR265:14, AR219:14,AR175:13, AR218:13, AR285:12, AR280:12, AR182:12, AR268:12, AR293:12,AR213:12, AR052:12, AR292:11, AR290:11, AR286:11, AR267:11, AR277:11,AR289:11, AR315:11, AR296:11, AR256:11, AR295:11, AR177:10, AR291:10,AR269:10, AR271:10, AR284:10, AR096:9, AR243:9, AR270:9, AR299:9,AR283:9, AR249:9, AR300:9, AR033:9, AR253:9, AR238:9, AR184:8, AR179:8,AR248:8, AR231:8, AR298:8, AR234:8, AR061:8, AR226:8, AR282:8, AR232:8,AR229:8, AR316:8, AR258:8, AR259:7, AR233:7, AR240:7, AR186:7, AR294:7,AR185:7, AR198:7, AR237:7, AR275:6, AR281:6, AR039:6, AR192:6, AR227:6,AR089:6, AR104:6, AR246:6, AR055:6, AR244:6, AR202:5, AR204:5, AR060:5,AR206:4, AR205:4, AR241:4, AR194:1 L0659:6, H0556:4, H0521:4, L0439:4,L0745:4, L0759:4, H0657:3, S0360:3, L0761:3, L0662:3, L0766:3, L0809:3,H0549:2, H0392:2, H0253:2, H0581:2, H0620:2, H0051:2, H0551:2, H0494:2,L0770:2, L0794:2, L0649:2, L0665:2, H0520:2, S0032:2, L0741:2, L0743:2,L0748:2, L0747:2, L0779:2, L0758:2, L0605:2, H0650:1, H0484:1, H0254:1,H0402:1, S0358:1, H0580:1, H0741:1, S0007:1, S0132:1, S0476:1, H0393:1,H0369:1, H0550:1, H0409:1, H0256:1, H0250:1, H0042:1, H0036:1, H0318:1,S0049:1, H0050:1, H0014:1, H0375:1, S6028:1, H0266:1, H0292:1, H0428:1,H0622:1, H0031:1, H0617:1, L0456:1, H0135:1, H0040:1, H0379:1, H0264:1,H0056:1, H0623:1, H0100:1, H0633:1, S0002:1, H0529:1, L0762:1, L5575:1,L0772:1, L0646:1, L0771:1, L0773:1, L0767:1, L0768:1, L0803:1, L0805:1,L0653:1, L5622:1, L4501:1, L0666:1, H0689:1, H0690:1, H0682:1, H0670:1,H0522:1, S0044:1, H0436:1, S0027:1, L0754:1, L0749:1, L0753:1, L0731:1,S0436:1, H0653:1, S0192:1, H0542:1, H0543:1, H0423:1 and S0424:1. 159HTLDU78 637702 169 L0758:3, H0253:1 and L0779:1. 160 HTLFA13 535937 170AR313:11, AR089:10, AR039:9, AR096:8, AR299:8, AR282:7, AR277:7,AR283:7, AR104:7, AR219:7, AR060:7, AR270:7, AR316:6, AR218:6, AR310:6,AR300:6, AR183:5, AR233:5, AR294:5, AR185:5, AR237:5, AR226:5, AR055:5,AR238:5, AR231:4, AR227:4, AR296:4, AR251:4, AR312:4, AR240:4, AR033:4,AR269:4, AR290:3, AR285:3, AR052:3, AR184:3, AR295:3, AR258:3, AR186:3,AR292:3, AR274:2, AR205:2, AR179:2, AR053:2, AR061:2, AR206:2, AR309:2,AR293:2, AR266:2, AR273:2, AR259:1, AR194:1, AR234:1, AR182:1, AR232:1,AR241:1, AR284:1 H0253:2 and S0011:1. 161 HTLGI89 835069 171 AR283:67,AR277:61, AR219:56, AR282:51, AR104:48, AR240:47, AR316:46, AR218:45,AR313:44, AR089:42, AR096:40, AR185:37, AR299:36, AR055:34, AR300:32,AR039:32, AR060:31 L0758:16, L0748:10, H0620:7, L0731:6, H0246:5,S0007:4, H0253:4, L0769:4, L0754:4, H0052:3, H0100:3, L0638:3, L5575:3,L0766:3, L0650:3, L0774:3, S0152:3, S3014:3, L0439:3, H0265:2, H0556:2,T0002:2, S6024:2, H0656:2, H0341:2, S0212:2, S0376:2, H0619:2, H0261:2,S0222:2, H0318:2, H0196:2, H0012:2, T0010:2, H0068:2, H0551:2, H0413:2,H04942, L0770:2, L5565:2, L3905:2, L0768:2, L0776:2, S3012:2, L0741:2,L0749:2, L0750:2, L0759:2, S0434:2, L0608:2, L0595:2, H0352:2, S0040:1,L0760:1, S0116:1, S0282:1, H0638:1, S0418:1, S0356:1, S0444:1, H0730:1,H0747:1, S0476:1, H0393;1, H0549:1, H0550:1, H0592:1, H0333:1, H0486:1,T0114:1, H0250:1, H0069:1, S0280:1. H0156:1, F10599:1, H0575:1, H0036:1,H0618:1, H0597:1, H0178:1, N0006:1, H0563:1, H0197:1, H0199:1, H0051:1,H0083:1, H0060:1, H0188:1, H0290:1, H0284:1, H0428:1, H0622:1, L0483:1,H0124:1, H0135:1, H0163:1, H0040:1, H0264:1, H0412:1, L0564:1, H0130:1,H0641:1, S0144:1, S0002:1, L0763:1, L0761:1, L0372:1, L0643:1, L0764:1,L0771:1, L0648:1, L0767:1, L0803:1, L0804:1, L0375:1, L0378:1, L0659:1,L0544:1, L0665:1, H0703:1, L0352:1, H0670:1, S0328:1, S0330:1, H0753:1,H0522:1, H0134:1, S0027:1, L0747:1, L0756:1, L0777:1, L0753:1, S0260:1,H0445:1, S0436:1, L0597:1, H0653:1 and S0194:1. 162 HTNBK13 831967 172L0779:5, L0731:4, L0593:4, H0046:3, L0776:3, L0666:3, H0031:2, L0772:2,L0774:2, L0805:2, H0670:2, L0439:2, L0754:2, L0777:2, L0758:2, L0590:2,T0002:1, L0717:1, H0632:1, L0622:1, T0082:1, H0581:1, H0263:1, T0115:1,H0597:1, L0471:1, H0012:1, H0620:1, H0163:1, T0067:1, L0770:1, L0637:1,L0388:1, L0657:1, L0382:1, L0664:1, S0126:1, H0660:1, S0378:1, H0521:1,L0747:1, L0750:1, L0756:1, L0752:1, L0755:1, L0759:1, S0031:1, L0599:1and L0603:1. 163 HTOAM11 664508 173 AR313:30, AR039:27, AR185:18,AR299:16, AR300:13, AR277:13, AR096:13, AR089:12, AR218:11, AR219:11,AR316:9, AR240:9, AR104:8, AR060:7, AR055:6, AR282:6, AR283:3 S0010:1and H0264:1. 164 HTOHO21 732808 174 H0556:3 and H0264:1. 165 HTPCO75853645 175 AR104:12, AR219:11, AR089:9, AR039:9, AR282:8, AR218:8,AR313:8, AR060:8, AR300:8, AR055:7, AR316:7, AR277:7, AR299:7, AR096:7,AR185:6, AR240:5, AR283:4 H0039:5, L0756:5, S0448:3, L0805:3, L0759:3,H0265:2, S0354:2, L0471:2, H0674:2, S0422:2, L0794:2, L0517:2, L0666:2,L0779:2, L0777:2, L0758:2, H0170:1, H0556:1, S0342:1, S0134:1, H0637:1,H0599:1, H0318:1, H0263:1, H0596:1, H0252:1, H0428:1, H0673:1, H0040:1,H0264:1, H0268:1, H0773:1, H0538:1, L0764:1, L0768:1, L0766:1, L0804:1,L0774:1, L0652:1, L0527:1, L0809:1, L0519:1, L0791:1, H0144:1, H0520:1,H0519:1, H0689:1, H0648:1, S0028:1, L0439:1, L0731:1, H0444:1, H0445:1,S0434:1, S0026:1, S0242:1 and H0543:1. 166 HTSFJ32 637720 176 AR104:9,AR039:7, AR277:5, AR282:4, AR313:4, AR299:4, AR240:3, AR089:3, AR283:3,AR300:3, AR096:3, AR185:3, AR055:2, AR316:2, AR219:2, AR218:2, AR060:2H0556:1, S0114:1, H0087:1, H0538:1, H0695:1 and L0774:1. 167 HTTCB60853401 177 L0794:11, L0809:11, L0750:9, L0805:8, L0791:7, L0747:7,L0800:6, L0758:6, L0759:6, H0620:5, L0749:5, S0358:4, H0135:4, L0769:4,L0659:4, L0780:4, H0556:3, L0471:3, H0040:3, L0804:3, S0360:2, H0393:2,H0550:2, H0592:2, H0333:2, S0049:2, H0124:2, S0438:2, L0771:2, L0662:2,L0803:2, L4501:2, H0547:2, L3832:2, L0779:2, L0755:2, L0731:2, S0434:2,L0603:2, H0506:2, H0713:1, H0717:1, H0294:1, H0662:1, H0729:1, H0734:1,S0045:1, H0607:1, H0586:1, H0587:1, L3816:1, T0040:1, L3653:1, S0280:1,H0590:1, S0010:1, H0581:1, H0251:1, H0041:1, H0565:1, H0570:1, H0123:1,H0081:1, H0050:1, H0188:1, H0039:1, H0622:1, H0038:1, H0063:1, H0412:1,H0413:1, S0440:1, S0210:1, S0002:1, L0763:1, L0770:1, L3905:1, L0761:1,L0641:1, L0768:1, L0766:1, L0375:1, L0806:1, L0776:1, L0789:1, L0790:1,L0666:1, L0663:1, L0665:1, L2258:1, L2654:1, H0520:1, H0660:1, H0672:1,H0539:1, S0380:1, H0521:1, H0696:1, H0555:1, L0744:1, L0748:1, L0757:1,H0445:1, L0584:1, L0589:1, S0242:1, S0194:1, H0008:1 and H0352:1. 168HTTEE41 840950 178 AR219:84, AR218:59, AR316:43, AR313:32, AR104:24,AR089:24, AR185:24, AR039:23, AR096:23, AR299:21, AR055:20, AR060:17,AR282:14, AR300:14, AR283:11, AR240:11, AR277:10 H0040:17, H0251:14,L0758:10, L0748:8, L0731:8, H0494:7, L0666:7, H0144:7, H0659:7, L0747:7,L0749:7, L0757:7, H0038:6, H0529:6, L0770:6, L0662:6, L0659:6, H0013:5,H0318:5, H0616:5, S0440:5, L0775:5, L0776:5, H0519:5, L0588:5, L0592:5,H0341:4, S0360:4, H0412:4, L0663:4, H0547:4, L0754:4, L0595:4, H0542:4,H0543:4, H0423:4, H0171:3, H0657:3, H0656:3, S0045:3, L3388:3, H0581:3,S0049:3, T0110:3, H0046:3, H0090:3, H0591:3, H0551:3, H0100:3, H0022:3,H0625:3, H0633:3, S0422:3. L0375:3, L0664:3, H0682:3, S0406:3, L0740:3,H0556:2, H0241:2, H0638:2, S0418:2, L0005:2, S0442:2, S0376:2, H0722:2,H0393:2. L0717:2, S0222:2, H0574:2, H0486:2, T0040:2, L0471:2, S0051:2,S0003:2, H0252:2, L0483:2, T0006:2, H0031:2, H0032:2, H0124:2, H0634:2,H0264:2, T0042:2, S0150:2, H0646:2, L0763:2, L0637:2, L0646:2, L0374:2,L0764:2, L0768:2, L0653:2, L0665:2, H0593:2, H0435:2, H0658:2, H0536:2,S0152:2, L3832:2, H0521:2, S3014:2, S0027:2, S0028:2, L0439:2, L0750:2,L0777:2, S0436:2, L0596:2, L0608:2, L0604:2, L0594:2, L0362:2, 50026:2,H0667:2, S0452:2, H0506:2, L0411:1, H0624:1, H0170:1, H0395:1, H0265:1,T0002:1, H0220:1, H0140:1, H0159:1, H0686:1, H0583:1, H0650:1, S0212:1,H0484:1, H0664:1, L0481:1, S0356:1, S0354:1, S0358:1, S0444:1, S0408:1,L3649:1, H0580:1, H0747:1, H0437:1, H0431:1, T0104:1, H0600:1, H0592:1,H0586:1, L3817:1, H0642:1, H0632:1, L2482:1, T0114:1, H0244:1, H0250:1,H0069:1, H0156:1, L0021:1, H0599:1, H0036:1, S0346:1, H0596:1, H0544:1,H0009:1, N0006:1, L0157:1, H0569:1, H0123:1, H0242:1, H0024:1, H0083:1,H0375:1, H0328:1, H0615:1, H0428:1, H0039:1, H0622:1, H0213:1, H0553:1,L0142:1, H0628:1, H0674:1, H0388:1, L0456:1, H0708:1, H0068:1, H0598:1,S0036:1, H0135:1, H0087:1, H0380:1, H0413:1, H0056:1, L0351:1, T0041:1,H0334:1, H0561:1, H0366:1, S0448:1, S0294:1, H0130:1, H0641:1, H0649:1,S0208:1, S0002:1, S0426:1, L0520:1, L0631:1, L0769:1, L0638:1, L5565:1,L0667:1, L0772:1, L0372:1, L0641:1, L0626:1, L0794:1, L0766:1, L0381:1,L0650:1, L0651:1, L0806:1, L0655:1, L0807:1, L0657:1, L0636:1, L0518:1,L0782:1, L0382:1, L0809:1, L3391:1, L2263:1, L2259:1, L2262:1, L0565:1,H0693:1, L3827:1, H0520:1, S0126:1, H0689:1, H0670:1, H0660:1, H0666:1,H0648:1, L0602:1, H0710:1, H0518:1, S0176:1, H0134:1, H0555:1, H0436:1,H0478:1, H0631:1, L0779:1, L0752:1, S0434:1, L0605:1, L0591:1, L0599:1,H0665:1, S0196:1, L2368:1, H0008:1 and H0352:1. 169 HTWEH94 561680 179AR313:9, AR039:7, AR096:5, AR300:3, AR185:3, AR089:3, AR299:3, AR277:2,AR316:2, AR240:2, AR060:2, AR104:2, AR218:1, AR282:1, AR055:1 L0766:1and H0436:1. 170 HTXFA72 853410 180 AR313:47, AR039:45, AR299:26,AR089:23, AR096:23, AR185:22, AR300:20, AR277:19, AR219:17, AR316:16,AR240:14, AR104:14, AR060:12, AR218:11, AR282:10, AR055:8, AR283:5H0265:1 171 HTXKF95 834438 181 AR266:6, AR309:6, AR178:6, AR176:5,AR162:5, AR161:5, AR163:5, AR282:5, AR096:5, AR312:5, AR104:5, AR053:5,AR271:4, AR185:4, AR060:4, AR055:4, AR183:4, AR308:4, AR246:4, AR168:4,AR181:4, AR269:4, AR229:4, AR192:4, AR263:4, AR175:4, AR274:4, AR193:4,AR225:4, AR165:4, AR267:4, AR182:4, AR164:4, AR316:4, AR213:4, AR228:4,AR166:4, AR242:3, AR240:3, AR270:3, AR272:3, AR212:3, AR264:3, AR283:3,AR243:3, AR275:3, AR313:3, AR179:3, AR235:3, AR268:3, AR239:3, AR293:3,AR237:3, AR171:3, AR180:3, AR289:3, AR173:3, AR215:3, AR172:3, AR089:3,AR231:3, AR210:2, AR061:2, AR290:2, AR230:2, AR201:2, AR233:2, AR204:2,AR196:2, AR277:2, AR223:2, AR226:2, AR299:2, AR177:2, AR190:2, AR247:2,AR300:2, AR296:2, AR257:2, AR285:2, AR222:2, AR033:2, AR200:2, AR039:2,AR191:2, AR311:2, AR199:2, AR297:2, AR291:2, AR288:2, AR203:2, AR227:2,AR232:2, AR188:2, AR294:2, AR236:2, AR255:2, AR189:2, AR286:2, AR260:2,AR238:2, AR211:2, AR174:2, AR287:1, AR234:1, AR261:1, AR256:1, AR216:1,AR262:1, AR252:1 L0754:41, L0747:8, L0755:5, L0659:4, H0265:2, H0556:2,H0586:2, L0471:2, H0553:2, L0764:2, L0662:2, L0794:2, L0748:2, L0751:2,L0749:2, L0750:2, H0305:1, S0358:1, S0046:1, H0441:1, H0599:1, H0569:1,H0050:1, H0051:1, H0030:1, H0124:1, H0616:1, L0770:1, L0769:1, L0800:1,L0644:1, L0363:1, L0803:1, L0804:1, L0775:1, L0806:1, L0783:1, L0666:1,L0665:1, HOl44:1, H0555:1, S3012:1, L0779:1, L0731:1, L0605:1, L0599:1,L0603:1, H0543:1, H0422:1 and H0506:1. 172 HUKDF20 566823 182 AR055:7,AR218:6, AR060:6, AR300:5, AR282:4, AR104:4, AR313:4, AR283:4, AR185:4,AR299:4, AR277:3, AR219:3, AR089:3, AR316:3, AR039:3, AR240:3, AR096:2H0261:1, H0266:1 and H0059:1. 173 HUSCJ14 894699 183 AR239:10, AR228:10,AR227:9, AR237:9, AR230:8, AR233:8, AR287:8, AR203:7, AR288:7, AR176:6,AR184:6, AR199:6, AR229:6, AR215:6, AR190:6, AR200:5, AR245:5, AR174:5,AR234:5, AR191:5, AR180:4, AR297:4, AR232:4, AR226:4, AR289:4, AR298:4,AR194:4, AR170:4, AR257:4, AR061:3, AR292:3, AR173:3, AR231:3, AR262:3,AR242:3, AR284:3, AR286:3, AR251:3, AR179:3, AR236:3, AR238:3, AR255:3,AR161:3, AR189:3, AR162:3, AR235:3, AR293:3, AR282:3, AR188:3, AR294:3,AR165:3, AR163:3, AR164:3, AR166:3, AR285:2, AR201:2, AR181:2, AR295:2,AR177:2, AR247:2, AR290:2, AR205:2, AR300:2, AR225:2, AR260:2, AR198:2,AR261:2, AR193:2, AR291:2, AR268:2, AR175:2, AR270:2, AR183:2, AR211:2,AR296:2, AR196:2, AR185:2, AR258:2, AR250:2, AR240:2, AR178:2, AR204:2,AR195:2, AR060:2, AR312:1, AR311:1, AR210:1, AR224:1, AR243:1, AR299:1,AR269:1, AR316:1, AR275:1, AR186:1, AR172:1, AR039:1, AR267:1, AR256:1,AR263:1, AR055:1, AR089:1, AR217:1 L2654:6, L0741:4, S0192:4, H0677:4,H0556:3, H0013:3, H0052:3, L0766:3, L0744:3, L0439:3, L0757:3, H0265:2,S0040:2, S0410:2, H0599:2, H0545:2, H0266:2, H0030:2, H0135:2, L3905:2,L5622:2, H0520:2, H0547:2, H0519:2, L0748:2, L0756:2, L0777:2, L0780:2,L0758:2, L0485:2, L0604:2, H0739:1, H0713:1, S0134:1, S0218:1, H0656:1,L2909:1, S0212:1, H0663:1, S0420:1, L1562:1, S0360:1, S0408:1, H0742:1,S0132:1, S0476:1, H0393:1, H0587:1, T0040:1, H0575:1, H0309:1, H0009:1,L0471:1, H0620:1, H0510:1, H0290:1, S0250:1, S0022:1, T0023:1, H0488:1,H0268:1, T0041:1, T0042:1, H0538:1, S0210:1, L0763:1, L0800:1, L0771:1,L0794:1, L0804:1, L0774:1, L0775:1, L5623:1, L0793:1, L2652:1, L2257:1,L2260:1, L0710:1, L2262:1, H0144:1, H0593:1, H0435:1, H0521:1, H0555:1,L0743:1, L0754:1, L0779:1, L0752:1, S0031:1, S0436:1, L0596:1, L0605:1,L0601:1, S0106:1, H0667:1, S0276:1 and L3576:1. 174 HUVDJ48 564853 184AR055:6, AR060:5, AR283:5, AR039:5, AR185:4, AR096:4, AR240:4, AR104:4,AR299:4, AR300:3, AR089:3, AR316:3, AR313:3, AR282:3, AR218:2, AR277:2,AR219:2 H0393:1, H0056:1 and L0662:1. 175 HBDAB91 864374 185 AR282:3,AR219:1 H0551:2, L0803:2, L0439:2, L0750:2, S0308:2, L0644:1, L0655:1,L0809:1, L0780:1 and L0752:1. HBDAB91 789532 193 176 HELGG84 851137 186AR218:39, AR219:33, AR299:7, AR104:6, AR055:5, AR313:4, AR300:4,AR185:4, AR060:4, AR240:3, AR282:3, AR089:3, AR316:2, AR283:2, AR039:2,AR277:2, AR096:1 L0750:5, S0045:2, S0212:1, H0742:1, S0300:1, S0010:1,H0505:1, S0049:1, H0266:1, L0598:1, L0662:1, L0809:1, S0374:1, H0696:1,L0758:1 and S0434:1. HELGG84 674456 194 177 HILCA24 869856 187 AR316:4,AR282:2, AR096:1, AR299:1, AR039:1 L0748:4, H0090:2, L0659:2, H0521:2,L0777:2, L0608:2, H0543:2, T0002:1, S0114:1, L3658:1, S0358:1, S0408:1,L3649:1, T0109:1, H0581:1, H0622:1, H0031:1, H0644:1, S0002:1, L0657:1,L0526:1, L0789:1, L0664:1, S0380:1, H0522:1, L0749:1 and L0779:1.HILCA24 782450 195 178 HYABC84 865064 188 AR313:19, AR219:16, AR218:13,AR104:13, AR096:11, AR316:11, AR240:10, AR299:10, AR089:10, AR282:9,AR185:9, AR039:9, AR283:8, AR277:7, AR060:6, AR055:5, AR300:5 HYABC84789854 196 179 HMCAZ04 668249 189 AR219:29, AR218:27, AR240:25,AR039:21, AR316:20, AR096:16, AR089: 15, AR299:13, AR283:13, AR282:12,AR060:9, AR313:9, AR055:8, AR300:8, AR277:7, AR185:6, AR104:4 S0410:22,S0408:18, S0476:15, S0132:14, H0584:9, S0358:9, S0002:8, H0521:8,L3388:7, L0748:7, S0442:6, H0494:6, L0599:6, S0142:5, L0777:5, S0278:4,L0483:4, L0775:4, L0659:4, H0543:4, S0444:3, H0733:3, H0046:3, H0284:3,H0039:3, H0674:3, H0591:3, H0641:3, S0144:3, L0771:3, L0773:3, S0374:3,L0439:3, H0422:3, H0677:3, H0556:2, T0002:2, H0657:2, S0212:2, S0360:2,H0574:2, H0486:2, H0635:2, H0575:2, H0231:2, H0024:2, H0286:2, H0622:2,H0644:2, H0673:2, S0440:2, H0633:2, S0426:2, L0764:2, L0766:2, L0774:2,L0651:2, L0655:2, L0664:2, H0593:2, H0658:2, H0710:2, S0044:2, S0404:2,L0745:2, L0747:2, L0731:2, S0434:2, L0581:2, S0011:2, S0276:2, H0423:2,H0506:2, H0171:1, H0167:1, H0713:1, H0716:1, H0656:1, S0298:1, H0662:1,H0459:1, H0638:1, S0348:1, S0354:1, S0376:1, H0580:1, H0729:1, H0742:1,H0722:1, H0734:1, H0208:1, S0045:1, H0632:1, H0075:1, H0156:1, H0042:1,H0036:1, H0318:1, H0581:1, H0251:1, H0309:1, H0545:1, H0107:1, H0083:1,H0179:1, H0687:1, H0292:1, S0214:1, H0031:1, H0628:1, H0617:1, L0055:1,H0032:1, H0316:1, H0090:1, H0038:1, H0040:1, H0063:1, T0067:1, H0264:1,L0564:1, H0202:1, S0014:1, H0560:1, S0372:1, H0649:1, S0344:1, L0640:1,L0371:1, L0770:1, L0667:1, L0765:1, L0803:1, L0376:1, L0805:1, L0653:1,L0542:1, L0783:1, L0809:1, L0663:1, H0701:1, S0126:1, H0689:1, H0672:1,S0328:1, H0539:1, L0602:1, H0522:1, S0406:1, H0187:1, H0727:1, S0028:1,S0206:1, L0743:1, L0756:1, L0779:1, L0752:1, L0759:1, S0308:1, H0343:1,S0436:1, L0485:1, L0601:1, H0653:1, S0196:1 and H0542:1. HMCAZ04 887445197 HMCAZ04 867910 198 HMCAZ04 858210 199 HMCAZ04 839783 200 180 HE8FD92901142 190 AR055:6, AR299:6, AR060:6, AR240:5, AR218:5, AR219:5,AR300:5, AR185:5, AR089:4, AR316:3, AR096:3, AR104:3, AR039:3, AR277:3,AR283:2, AR282:2, AR313:2 HE8FD92 888274 201 HE8FD92 869847 202 HE8FD92856544 203 HE8FD92 843825 204

Table 1C summarizes additional polynucleotides encompassed by theinvention (including cDNA clones related to the sequences (Clone ID:),contig sequences (contig identifier (Contig ID:) contig nucleotidesequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ IDNO:B). The first column provides a unique clone identifier, “Clone ID:”,for a cDNA clone related to each contig sequence. The second columnprovides the sequence identifier, “SEQ ID NO:X”, for each contigsequence. The third column provides a unique contig identifier, “ContigID:” for each contig sequence. The fourth column, provides a BACidentifier “BAC ID NO:A” for the BAC clone referenced in thecorresponding row of the table. The fifth column provides the nucleotidesequence identifier, “SEQ ID NO:B” for a fragment of the BAC cloneidentified in column four of the corresponding row of the table. Thesixth column, “Exon From-To”, provides the location (i.e., nucleotideposition numbers) within the polynucleotide sequence of SEQ ID NO:Bwhich delineate certain polynucleotides of the invention that are alsoexemplary members of polynucleotide sequences that encode polypeptidesof the invention (e.g., polypeptides containing amino acid sequencesencoded by the polynucleotide sequences delineated in column six, andfragments and variants thereof). TABLE 1C cDNA Clone SEQ ID CONTIG BACID: SEQ ID EXON ID NO: X ID: A NO: B From-To H6EEU40 12 757048 AC026285399  1-414  440-1918 H6EEU40 12 757048 AC023920 400  1-416  442-1920H6EEU40 12 757048 AC026285 401   1-1458 1729-1836 H6EEU40 12 757048AC023920 402   1-1459 1730-1837 HACAB68 13 584773 AL160283 403   1-2811HACAB68 13 584773 AL354793 404   1-3734 3843-4723 HACAB68 13 584773AL356058 405   1-3055 3165-4045 HACBJ56 14 847112 AC069497 406   1-1172470-3367 4908-5262 5641-5756 7886-8200  9815-11138 HACBJ56 14 847112AC007104 407  1-802 2342-2695 3074-3189 5319-5633 7248-8571 HACBJ56 14847112 AC069497 408  1-453 HACBJ56 14 847112 AC007104 409  1-453 HADMB1515 847116 AC026666 410  1-385 406-780 HADMB15 15 847116 AC026281 411 1-114 430-875  896-1262 HAGDW20 16 637489 AC006453 412   1-1568 HAGDW2016 637489 AC005629 413   1-1569 HAGDW20 16 637489 AC010098 414   1-1569HAGDW20 16 637489 AC006453 415  1-438 HAGDW20 16 637489 AC006453 416 1-375 HAGDW20 16 637489 AC005629 417  1-438 HAGDW20 16 637489 AC005629418  1-375 HAJCH70 19 827275 AL159987 419   1-2168 HATCD80 21 826098AL158801 420   1-1974 HATCD80 21 826098 AL158801 421  1-90 HATCI03 22580805 AL137119 422  1-81 824-941  972-1185 2432-2705 3880-48124880-5011 5828-6591 8231-8398 8618-8767 9466-9728 HATCI03 22 580805AL138688 423  1-81 825-942  973-1186 2433-2706 3881-4795 4870-50015818-6581 8221-8388 8608-8757 9456-9718 HATCI03 22 580805 AL137119 424 1-542 HATCI03 22 580805 AL138688 425  1-542 HBAGD86 23 838799 AC016755426  1-41 1648-1993 2035-3552 3554-6713 HBAGD86 23 838799 AC016755 427 1-161 696-809 2256-2753 6910-6991 7733-7857 9267-9458 10650-1073411114-11562 11678-11801 12524-12817 14494-15914 HBAGD86 23 838799AC016755 428  1-217 HBHAA05 24 603174 AL353743 429  1-677 HBHAA05 24603174 AL161453 430  1-677 HBHAA05 24 603174 AL161453 431  1-339 HBHAA8125 846465 AC006059 432  1-230 1619-1699 1953-2090 2986-3054 3665-37863902-4406 4457-4674 5129-5531 5660-5811 5934-5969 7563-7959 8086-91959591-9735 ‘9788-10149 HBHAA81 25 846465 AC018471 433  1-230 1619-16991965-2090 2986-3054 3665-3786 3902-4405 4456-4673 5128-5530 5659-58105933-5968 7561-7957 8084-9193 9589-9733  9786-10146 HBHAA81 25 846465AC006059 434  1-340 501-802 HBHAA81 25 846465 AC006059 435  1-6611538-1684 3489-3680 3832-3933 4241-4410 5782-5872 5998-6150 HBHAA81 25846465 AC018471 436  1-661 1539-1672 HBHAA81 25 846465 AC018471 437 1-340 501-802 HBIAA59 26 806303 AL121929 438   1-1114 1186-3742 HBIAA5926 806303 AL121929 439  1-226 HBIAA59 26 806303 AL121929 440  1-243HBJAC40 27 841235 AC007606 441  1-89 520-616 811-972 1604-1874 1982-50385125-5260 6459-6956 7370-7473 7507-7774 8952-9321 HBJAC40 27 841235AC007606 442  1-403  428-1236 HBJDW56 29 520401 AC005532 443  1-626HBJDW56 29 520401 AC005532 444  1-516 HBJDW56 29 520401 AC005532 445 1-176 HCDCY76 37 837972 AP001528 446   1-3072 HCDCY76 37 837972AP001528 447  1-380 HCEEU18 39 688041 AC008469 448  1-169 HCEEU18 39688041 AC026400 449  1-170 HCEEU18 39 688041 AC008469 450  1-304 420-6021427-2108 2323-2645 3613-3987 4129-4442 4600-4731 4868-5039 5408-55385624-5776 6317-7734 HCEEU18 39 688041 AC008469 451  1-294 HCEEU18 39688041 AC026400 452  1-98 HCEEU18 39 688041 AC026400 453  1-407 HCEGX0540 827060 AL133227 454  1-32  712-1071 3453-3870 4197-4326 4639-47515131-5202 5588-5638 7454-8108 8670-8767 9511-9692  9754-1013411109-11226 12456-12607 15237-15316 18143-18311 18429-18478 20682-2098220988-21295 22686-23061 23358-23495 24076-24612 25196-25334 26760-2692627041-27152 27271-27379 27697-28289 29024-29340 29761-29840 31168-32681HCEGX05 40 827060 AL161661 455  1-130 443-555  935-1006 1392-14423258-3912 4474-4571 5315-5496 5558-5938 6915-7032 8262-8413 11042-1112113948-14116 14234-14283 16487-16787 16793-17100 18494-18869 19166-1930319884-20420 21002-21140 22566-22732 22847-22958 23077-23185 23503-2409524826-25142 25563-25642 26969-28482 HCEGX05 40 827060 AL133227 456  1-51476-521  842-1226 1375-1490 3745-4016 4046-4229 4430-4855 5300-60536598-6883 7406-7446 7461-8437 8550-8681 8888-8919 8943-9353 9458-9544 9834-10607 11550-11629 12196-12374 13532-14886 HCEGX05 40 827060AL161661 457  1-418 HCEGX05 40 827060 AL161661 458  1-50 475-520 841-1225 3744-4014 4044-4227 4428-4853 4925-5089 5298-6051 6649-6801HCFLN88 41 610000 AC005089 459  1-594 1779-2065 2224-2411 3295-35883962-4463 5317-5561 5835-6210 6750-7793 HCFLN88 41 610000 AC005089 460 1-141 HCFLN88 41 610000 AC005089 461  1-215 HCMSX51 43 788643 U96629462   1-3014 HCMSX51 43 788643 AC040975 463   1-3014 HCNCO11 44 775086AC011319 464  1-700 HCNCO11 44 775086 AC069204 465  1-700 HCNCO11 44775086 AC011319 466  1-354 HCNCO11 44 775086 AC011319 467  1-338 HCNCO1144 775086 AC069204 468  1-354 HCQBH72 46 637548 AC073530 469   1-1790HCQBH72 46 637548 AC073530 470  1-106 HCQBH72 46 637548 AC073530 471 1-410 HCWAE64 50 535893 AL157935 472   1-1319 2024-2316 2937-29843126-3281 5595-5703 5788-6574 6667-6733 6788-6880 6962-7303  8111-1186912019-12418 12420-12679 13140-13191 HCWAE64 50 535893 AL157935 473  1-1316 HCWAE64 50 535893 AL157935 474  1-309 HDPIY31 55 886159AL356790 475  1-114  949-1189 2041-2318 2541-2711 2797-2871 3152-7720HDP1Y31 55 886159 AL118506 476  1-241 1067-1317 1567-1737 1823-18972178-6746 HDP1Y31 55 886159 AL356790 477  1-104 116-297 HDPIY31 55886159 AL356790 478  1-481 HDP1Y31 55 886159 AL118506 479  1-89 HDPPQ3058 684292 AL022315 480  1-968 HDPPQ30 58 684292 AL022315 481  1-255HDTFX18 60 801957 AC013303 482  1-832 1218-1410 2344-2463 3663-39554307-4617 5925-6011 10329-10419 11011-11162 12512-12600 13752-1389414068-14375 14493-15033 15161-15932 HE2CM39 61 553651 AC018391 483  1-3570 3779-3904 4646-5979 6339-6701 6710-8473 HE2CM39 61 553651AC018391 484  1-438 HE2CM39 61 553651 AC018391 485   1-1402 1586-18712685-2797 3088-3503 4900-5170 5789-5882 6089-6195 HE2P093 63 771655AC020894 486  1-353 749-1198 2724-2986 4932-5578 7481-7617 8108-82578515-8849 9840-9968 10287-10827 11376-14474 14652-15073 15510-1708317304-20501 HE2P093 63 771655 AC008590 487  1-648 2551-2687 3178-33273585-3919 4910-5038 5357-5897  6446-10147 10584-12159 12380-15574HE2P093 63 771655 AC021468 488  1-353  749-1198 2724-2986 4934-55797482-7618 8109-8258 8516-8850 9841-9969 10288-10828 11377-1362713631-13748 13762-15078 15515-17088 17309-20507 HB2P093 63 771655AC020894 489  1-372 HE2P093 63 771655 AC020894 490  1-315  893-1242HE2P093 63 771655 AC021468 491  1-350 HE2P093 63 771655 AC021468 492 1-372 HE6FU11 64 827236 AL021578 493  1-116 2674-2776 3489-40637279-7402  9706-10120 10217-10368 12042-12219 12315-12924 14271-1438014463-14842 16153-16301 HE6FV29 65 588454 AL162401 494   1-1425 HEBFR4669 847064 AC006483 495  1-70 282-644  789-4243 HEBFR46 69 847064AC073481 496   1-2167 2174-3461 HEBFR46 69 847064 AC006483 497  1-344HEBFR46 69 847064 AC006483 498  1-195 HEBGE07 70 798096 AC021918 499  1-1899 HEBGE07 70 798096 AC021918 500  1-225 HEGAU15 71 834379AC009404 501   1-1121 HEGAU15 71 834379 AC011638 502   1-1119 HEGAU15 71834379 AC009404 503  1-363 HEGAU15 71 834379 AC009404 504  1-446 HEGAU1571 834379 AC011638 505  1-446 HEGAU15 71 834379 AC011638 506  1-363HFCEI04 73 692438 AC068996 507  1-865 HFCEI04 73 692438 AC068303 508 1-865 HFCFE20 74 701985 AC044815 509  1-746 1575-2050 2508-32934818-5412 6081-6547 6748-6935 7843-8192 8425-8581 9095-9217 9266-940711036-11432 12081-12179 13701-13787 13976-16347 HFCFE20 74 701985AC026587 510   1-2259 HFCFE20 74 701985 AL355175 511   1-2260 HFPDR62 76839400 AC024938 512   1-2651 HFPDS07 77 821646 AC067945 513   1-3965HFPDS07 77 821646 AC067945 514  1-814 HFPDS07 77 821646 AC067945 515 1-743 HFVHW43 78 570948 AL132795 516  1-253 1142-1455 1576-21502529-2966 4374-4471 4991-5361 6514-7738 7936-8053 9858-9979 11930-1210112401-12525 12531-12712 16593-16786 17053-17214 18919-19396 21174-2132721724-22296 22515-23071 HFVHW43 78 570948 AL132795 517   1-6181 HFVHW4378 570948 AL132795 518  1-287 622-861 HGBHP91 81 693011 AL356056 519  1-1048 HGBHP91 81 693011 AL136982 520   1-1048 HGBHP91 81 693011AL356056 521  1-238 HGBHP91 81 693011 AL356056 522  1-135 HGBHP91 81693011 AL136982 523  1-238 HGBHP91 81 693011 AL136982 524  1-136 HHEAK4582 765278 AL035690 525   1-2148 4277-4419 5252-5365 5452-6322 6863-7710HHEAK45 82 765278 AC010388 526   1-2149 HHEAK45 82 765278 AL035690 527 1-732 HHEAK45 82 765278 AL035690 528  1-86 175-566 HHFFS40 84 824059AC022423 529   1-2017 HHFFS40 84 824059 AC025178 530   1-2017 HHFFS40 84824059 AC022444 531   1-2017 HHPSA85 86 658695 AL354831 532  1-2912324-3412 HHPSA85 86 658695 AC018674 533  1-291 2324-3412 HHSBI06 87639097 AF285442 534   1-1170 1250-1439 1565-1850 2214-2632 HHSBI06 87639097 AF271897 535   1-1174 1253-1444 1568-1853 2217-2659 4394-48765269-6156 7228-8366 8574-8852 HHSBI06 87 639097 AC025857 536   1-11721254-1442 1566-1851 2215-2618 4423-4905 5298-6179 7253-8391 8599-8877HHSBI06 87 639097 AF285442 537  1-483 HHSBI06 87 639097 AF285442 538 1-323 420-676 774-935 1372-1637 HHSBI06 87 639097 AF271897 539  1-522HHSBI06 87 639097 AF271897 540  1-323 420-676 774-935 1372-1637 HHSBI0687 639097 AC025857 541  1-522 HHSBI06 87 639097 AC025857 542  1-323420-676 774-935 1372-1637 HHSBI65 88 801910 AF205589 543   1-17031798-2217 2302-3089 HHSBI65 88 801910 AF205589 544  1-531  571-17591862-2104 2219-2722 HHSGL28 89 801912 AC024242 545   1-2154 HHSGL28 89801912 AC020584 546  1-215  233-1205 HHSGL28 89 801912 AC024242 547 1-216  952-1969 HHSGL28 89 801912 AC020584 548  1-635 HISAT67 90 843549AC013403 549  1-753  852-1545 1734-1816 1930-2061 2259-2428 2573-26482685-2987 3135-4126 4242-4543 4732-4905 5033-5145 5298-5341 5530-57156059-6126 HISAT67 90 843549 AC013403 550  1-102 HKACI79 94 853361AC006512 551  1-658 3090-3543 4479-5105 5885-6846 7103-9707  9914-1029311523-12034 12067-12181 13769-14031 14199-14291 14584-14790 15123-1515417039-17482 17539-17987 18697-19052 19112-19380 20023-20268 21158-2159821817-22221 23565-23665 23906-24076 24981-25506 25510-25861 25981-2664526661-27449 27717-27812 27991-28024 28437-28888 29651-33442 33621-3408934245-34808 34819-35284 35854-35960 38525-38771 HKACI79 94 853361AC011841 552  1-710  902-1864 1997-2121 2334-3824 4232-5905 HKACI79 94853361 AC011043 553  1-712  904-1867 1874-1906 2000-2124 2337-3891HKACI79 94 853361 AC078939 554  1-646  837-1797 1804-1836 1930-38204161-5834 HKACI79 94 853361 AC006512 555  1-315 439-531  707-10801144-1227 1491-1845 2113-2321 2700-3556 3818-4307 4336-4813 4958-5775HKACI79 94 853361 AC006512 556  1-738 HKACI79 94 853361 AC011841 557 1-541 HKACI79 94 853361 AC011841 558  1-105 HKACI79 94 853361 AC011043559  1-105 HKACI79 94 853361 AC078939 560  1-564 HKAC179 94 853361AC078939 561  1-105 HKIXC44 95 716213 AC016240 562  1-195 476-552679-763 1040-1119 1998-3764 HKIXC44 95 716213 AF261720 563  1-195475-551 678-762 1039-1118 1998-3764 HKIXC44 95 716213 AC016240 564 1-423 HKIXC44 95 716213 AF261720 565  1-423 HKIXC44 95 716213 AF261720566  1-206 HKTAB41 96 695732 AC006451 567  1-737 HLHAP05 98 638476AC009097 568  1-101 HLHCS23 99 560663 AL356385 569   1-1419 HLHCS23 99560663 AC016501 570   1-1419 HLHCS23 99 560663 AL356385 571  1-560HLHCS23 99 560663 AC016501 572  1-560 HLMGP50 101 647603 AC019101 573  1-1039 HLMGP50 101 647603 AC01910I 574  1-100 HLMMX62 102 688051AL356320 575  1-275 HLMMX62 102 688051 AL356320 576  1-122 HLMMX62 102688051 AL356320 577  1-377 HLQCX36 103 584786 AC013758 578  1-316HLQCX36 103 584786 AC024953 579  1-303 HLQCX36 103 584786 AC018740 580 1-280  942-1052 HLQCX36 103 584786 AC016539 581  1-293 HLQCX36 103584786 AC012278 582  1-272 HLQCX36 103 584786 AC068854 583  1-183HLQCX36 103 584786 AC019205 584  1-149 HLQCX36 103 584786 AC011175 585 1-318 HLQCX36 103 584786 AL157366 586  1-140 HLQCX36 103 584786AC067890 587  1-223 HLQCX36 103 584786 AC032044 588  1-281 HLQCX36 103584786 AC016615 589  1-300 HLQCX36 103 584786 AC018753 590  1-205HLQCX36 103 584786 AC015900 591  1-167 HLQCX36 103 584786 AL162592 592 1-300 HLQCX36 103 584786 AC073568 593  1-226 HLQCX36 103 584786AC026967 594  1-305 HLQCX36 103 584786 AC026107 595  1-275 HLQCX36 103584786 AC011127 596   1-1365 HLQCX36 103 584786 AC034243 597  1-3122334-2364 HLQCX36 103 584786 AL356441 598  1-289 HLQCX36 103 584786AC027531 599  1-298 HLQCX36 103 584786 AC022950 600  1-311 HLQCX36 103584786 AC010958 601  1-131 HLQCX36 103 584786 AC010073 602  1-175HLQCX36 103 584786 AP001397 603   1-1365 HLQCX36 103 584786 AC055119 604 1-320 HLQCX36 103 584786 AC027105 605  1-308 HLQCX36 103 584786AC022795 606  1-300 HLQCX36 103 584786 AC018445 607  1-140 HLQCX36 103584786 AL358115 608  1-142 HLQCX36 103 584786 AL355975 609  1-322HLQCX36 103 584786 AL096841 610  1-281 HLQCX36 103 584786 AC027414 611 1-270 HLQCX36 103 584786 AC023008 612  1-296 HLQCX36 103 584786AL357125 613  1-278 HLQCX36 103 584786 AC073446 614  1-299 HLQCX36 103584786 AC015989 615   1-1365 HLQCX36 103 584786 AC020873 616  1-306HLQCX36 103 584786 AC068854 617   1-1087 HLQCX36 103 584786 AC018753 618 1-523 HLQCX36 103 584786 AC011127 619  1-686 HLQCX36 103 584786AP001397 620  1-98 HLQCX36 103 584786 AL358115 621   1-2327 HLQCX36 103584786 AC015989 622  1-685 HLQCX36 103 584786 AC015989 623  1-98 HLQCX36103 584786 AC020873 624  1-126 HLYDF73 106 566869 AL122127 625  1-583HMDAB29 109 584789 AC027264 626  1-147 HMDAB29 109 584789 AC068682 627 1-153 HMDAB29 109 584789 AL354887 628   1-1433 HMDAB29 109 584789AL157408 629   1-1434 HMDAB29 109 584789 AL354887 630  1-577 HMDAB29 109584789 AL354887 631  1-196 HMDAB29 109 584789 AL157408 632  1-577HMDAB29 109 584789 AL157408 633  1-196 HMDAD44 110 566854 AC012370 634 1-145 2813-4454 HMDAD44 110 566854 AC034121 635   1-1569 HMDAD44 110566854 AC012370 636  1-787 HMDAD44 110 566854 AC012370 637  1-622HMIBF07 112 603528 AC022833 638   1-1721 HMICP65 113 847403 AL162741 639 1-45 HMICP65 113 847403 AL162741 640  1-102 HMWBL03 117 822861 AC012052641  1-130 548-784 4520-4887 5112-6285 6741-6888 7577-7727 7951-8582 8927-10292 HMWBL03 117 822861 AC011667 642  1-138 1281-1681 2270-26323070-3372 3865-3990 4407-4644 8378-8745  8970-10143 10599-1074611435-11585 11809-12440 12785-14150 HMWBL03 117 822861 AC012052 643 1-303 HNFGRO8 118 825417 AC006369 644   1-1423 HNGAK51 119 603910AC013443 645  1-913 HNGAK51 119 603910 AC013443 646  1-406 HNGAK51 119603910 AC013443 647  1-297 HNGDX18 120 1145071 AL391069 648   1-1403HNGDX18 120 1145071 AL158846 649  1-193 208-577  894-1167 1401-16291918-3320 4039-4082  9400-10337 HNGDX18 120 1145071 AL391069 650  1-274HNGDX18 120 1145071 AL158846 651  1-117 HNGPJ2S 124 834942 AP002781 652  1-1472 HNHKV56 127 800877 AC009396 653   1-1605 HODBB70 129 520196AC006322 654  1-561 HODBB70 129 520196 AC073110 655  1-561 HODBB70 129520196 AC025553 656  1-561 HODBB70 129 520196 AC006322 657   1-1741HODBB70 129 520196 AC006322 658  1-354 HODBB70 129 520196 AC073110 659  1-1741 HODBB70 129 520196 AC073110 660  1-354 HORBS82 133 638293AL034419 661   1-1798 HORBS82 133 638293 AL034419 662   1-1186 HPFCI36137 855966 AL161652 663  1-174  313-4710 HPFD137 138 862056 AC000090 664 1-29 566-712 1355-1425 3075-3241 3725-3806 4295-4357 5382-55716510-7016 7981-8321 HPJCW58 140 612866 AC024735 665   1-1160 HRADF49 146866481 AC068946 666  1-142  359-1108 1191-1345 1445-2140 2314-29353040-3156 3395-4126 4311-4460 4749-5820 HRADF49 146 866481 AC060820 667 1-142 359-1109 1193-1348 1448-2142 2318-2944 3056-3166 3405-41364321-4472 4762-5836 HRADF49 146 866481 AC068946 668  1-812 1124-12631281-2283 2470-2572 2752-2935 3851-3974 4153-4548 4602-4810 4980-51115262-5346 5434-5498 5609-5695 5871-5930 6448-6487 HRADF49 146 866481AC060820 669  1-686 HRDDQ39 147 840405 AC009152 670  1-755 HROEA08 149866190 AC010894 671   1-3018 HROEA08 149 866190 AC010894 672  1-138HROEA08 149 866190 AC010894 673  1-299 HSAVW42 151 637660 AC021117 674 1-865 HSAVW42 151 637660 AC021117 675  1-336  397-651 HSAVW42 151637660 AC021117 676  1-185 HSLHX15 153 777861 AC072032 677  1-364HSLHX15 153 777861 AC002518 678  1-247 HSLHX15 153 777861 AC022305 679 1-686 HSLHX15 153 777861 AC078916 680  1-364 HSLHX15 153 777861AC072032 681  1-288 HSLHX15 153 777861 AC078916 682  1-288 HSSEF77 155658725 AC005041 683  1-68  87-493 711-838  997-1167 2227-2960 3326-46414768-5786 HSSEF77 155 658725 AC005041 684   1-2920 3439-3667 3839-4332HSSEF77 155 658725 AC005041 685  1-143 HT4FV41 156 853400 AC011547 686 1-170 793-936 2771-3041 3691-3788 5141-5252 5755-6030 6325-64077214-7551 8653-8940 9033-9136 9428-9907 11266-11659 12082-1226313451-13544 13664-13699 13769-13936 14571-14761 14897-14997 15135-17127HT4FV4I 156 853400 AC005331 687  1-88 224-324  462-2454 HT4FV41 156853400 AC023470 688  1-80 204-242 313-477 1213-1300 1436-1536 1673-3658HT4FV41 156 853400 AC005331 689  1-607 HT4FV41 156 853400 AC023470 690 1-606 HT5FX79 157 794169 AC020978 691   1-4351 4423-4590 4875-50615211-5413 5519-5726 5755-6138 6281-6319 6402-7114 7359-7460 7715-79188030-8144 8612-9037 9280-9760 HT5FX79 157 794169 AC020978 692  1-431HT5FX79 157 794169 AC020978 693  1-38 115-354 HTEDJ28 161 762845AC025974 694   1-2357 HTEDJ28 161 762845 AC013370 695   1-2357 HTEDS12162 838621 AC021491 696  1-124 290-781 1447-1562 1650-1767 2309-24173273-3466 3935-4120 5213-5358 6216-6605 7621-7744 8491-8761 9044-91759353-9523  9966-10843 11395-11839 HTEDS12 162 838621 AC021491 697  1-2283662-4225 HTEHA56 163 806461 AC008751 698  1-469 1023-1372 1542-16941724-3063 3371-3477 3651-3905 4073-4931 4999-6547 HTEHA56 163 806461AC009763 699   1-1487 HTEHA56 163 806461A C008749 700   1-1487 HTEHA56163 806461A C008751 701  1-575 HTEHA56 163 806461A C009763 702  1-577HTEHA56 163 806461A C009763 703  1-859 HTEHA56 163 806461A C008749 704 1-575 HTEHA56 163 806461A C008749 705  1-582 HTEJD29 164 695798AL354733 706   1-1292 HTEJD29 164 695798A C007943 707   1-1292 HTEJD29164 695798A L354733 708  1-184 HTEJD29 164 695798A L354733 709  1-591212-1284 1905-1956 2351-2840 4126-5105 5892-6298 6726-7122 7204-77137747-7932 HTHBZ06 167 832477 AC068768 710  1-835 HTLBT80 168 840045AL133227 711  1-51 476-521  842-1226 1375-1490 3745-4016 4046-42294430-4855 5300-6053 6598-6883 7406-7446 7461-8437 8550-8681 8888-89198943-9353 9458-9544  9834-10607 11550-11629 12196-12374 13532-14886HTLBT80 168 840045 AL133227 712  1-32  712-1071 3453-3870 4197-43264639-4751 5131-5202 5588-5638 7454-8108 8670-8767 9511-9692  9754-1013411109-11226 12456-12607 15237-15316 18143-18311 18429-18478 20682-2098220988-21295 22686-23061 23358-23495 24076-24612 25196-25334 26760-2692627041-27152 27271-27379 27697-28289 29024-29340 29761-29840 31168-32681HTLDU78 169 637702 AC011444 713   1-1305 HTLDU78 169 637702 AC011444 714 1-285 HTLDU78 169 637702 AC011444 715  1-274 HTLFA13 170 535937AC022007 716   1-1127 HTLFA13 170 535937 AC021995 717   1-1115 HTLFA13170 535937 AC007783 718   1-1144 HTLFA13 170 535937 AC022007 719  1-1729 HTLFA13 170 535937 AC022007 720  1-179 184-696 HTLFA13 170535937 AC021995 721  1-106 132-190 674-831 1456-1588 4811-4933 5118-5304HTLFA13 170 535937 AC021995 722  1-179 184-696 894-945 HTLFA13 170535937 AC007783 723  1-169 180-258 681-859  864-1376 3240-3503 HTLFA13170 535937 AC007783 724   1-1729 HTLGI89 171 835069 AC048342 725  1-130HTLGI89 171 835069 AC009453 726  1-143 HTLGI89 171 835069 AC022231 727 1-151 HTLGI89 171 835069 AC009524 728  1-151 HTLGI89 171 835069AC048342 729  1-118 HTOAM11 173 664508 AC002369 730  1-586 2559-26513329-3426 3756-5088 HTOAM11 173 664508 AP001486 731   1-1191 HTOAM11 173664508 AP000875 732   1-1192 HTOAM11 173 664508 AC002369 733  1-228HTOAM11 173 664508 AP001486 734  1-711 HTOAM11 173 664508 AP001486 735 1-374 HTOAM11 173 664508 AP000875 736  1-710 HTOHO21 174 732808AC022221 737  1-85 394-740  781-1562 1622-2429 3831-4082 4239-60537230-7365 8195-8379 11677-11990 12508-12710 HTOHO21 174 732808 AC007897738   1-1586 2763-2898 3728-3912 7210-7523 8041-8243 HTOHO21 174 732808AC022221 739  1-184 HTOHO21 174 732808 AC007897 740  1-184 HTSFJ32 176637720 AC015734 741  1-80 562-915  925-4400 HTSFJ32 176 637720 AC015734742  1-463 HTSFJ32 176 637720 AC015734 743  1-359 HTTEE41 178 840950AC018921 744  1-92 318-578 837-912 1091-1249 1321-1387 1862-21922485-2579 2708-2831 3685-4257 4547-5127 5811-6037 6562-7076 7541-76788069-8191 10100-10207 11102-11688 11721-11847 12201-12335 12532-1264112888-12991 13027-13546 13637-16146 HTTEE41 178 840950 AC018921 745 1-100 HTWEH94 179 561680 AC004858 746   1-1349 1370-1744 HTWEH94 179561680 AC004858 747  1-94 HTWEH94 179 561680 AC004858 748  1-199 HTXFA72180 853410 AP001812 749   1-1015 HTXFA72 180 853410 AP000822 750  1-1015 HTXFA72 180 853410 AP001812 751  1-130 HTXFA72 180 853410AP000822 752  1-527 HTXKF95 181 834438 AC004242 753  1-981 HTXKF95 181834438 AC008083 754  1-981 HTXKF95 181 834438 AC004242 755  1-984HTXKF95 181 834438 AC004242 756  1-118 HTXKF95 181 834438 AC008083 757 1-984 HTXKF95 181 834438 AC008083 758  1-173 HUSCJ14 183 894699AC007040 759  1-149 394-889 1061-1139 2097-2249 2852-3007 5021-50895217-5919 6119-8896 HUSCJ14 183 894699 AC007040 760  1-854 HUSCJ14 183894699 AC007040 761  1-397 HELGG84 186 851137 AC025019 762   1-2121  1-2121 HELGG84 186 851137 AC012202 763   1-2122   1-2122 HELGG84 186851137 AC025019 764  1-573  1-573 HELGG84 186 851137 AC025019 765  1-401158-2410  1-40 1158-2410 HELGG84 186 851137 AC012202 766  1-573  1-573HYABC84 188 865064 AL132825 767   1-2512 2604-2740 2974-3241   1-25122604-2740 2974-3241 HYABC84 188 865064 AL132825 768  1-553  1-5531059-1263 1059-1263 3121-3476 3121-3476 5284-5734 5284-5734 6284-65136284-6513 6786-7426 6786-7426 8674-8733 8674-8733 10656-1093310656-10933 11453-11555 11453-11555 12991-13079 12991-13079 13839-1428113839-14281 14527-14827 14527-14827 15156-15685 15156-15685 15835-1604615835-16046 16166-16604 16166-16604 16736-19566 16736-19566 19658-1979419658-19794 20028-20295 20028-20295 HYABC84 188 865064 AL132825 769 1-188  1-188 HE8FD92 190 901142 AL359176 770   1-2410   1-2410   1-24102420-4226 2420-4226 2420-4226 HE8FD92 190 901142 AL139152 771  1-826 863-2063 2125-4935 4945-6753  1-826  863-2063 2125-4935 4945-6753 1-826  863-2063 2125-4935 4945-6753  1-826  863-2063 2125-49354945-6753 HE8FD92 190 901142 AL109937 772  1-168 233-930 1572-17482463-3391 HE8FD92 190 901142 AC027209 773   1-1201   1-1201   1-12011263-1531 1263-1531 1263-1531 1648-5860 1648-5860 1648-5860 HE8FD92 190901142 AL356004 774   1-1052 1062-2871 HE8FD92 190 901142 AL139152 775 1-560  760-1714 1740-3644 3736-4319 4872-4998  1-560  760-17141740-3644 3736-4319 4872-4998  1-560  760-1714 1740-3644 3736-43194872-4998  1-560  760-1714 1740-3644 3736-4319 4872-4998 HE8FD92 190901142 AL109937 776  1-437 HE8FD92 190 901142 AC027209 777  1-560  1-560 1-560  747-1721  747-1721  747-1721 1875-3650 1875-3650 1875-36503698-4325 3698-4325 3698-4325 4657-4693 4657-4693 4657-4693 4879-50084879-5008 4879-5008 HE8FD92 190 901142 AC027209 778  1-423  1-423  1-423HE8FD92 190 901142 AL356004 779  1-560

Table 1D: The polynucleotides or polypeptides, or agonists orantagonists of the present invention can be used in assays to test forone or more biological activities. If these polynucleotides andpolypeptides do exhibit activity in a particular assay, it is likelythat these molecules may be involved in the diseases associated with thebiological activity. Thus, the polynucleotides or polypeptides, oragonists or antagonists could be used to treat the associated disease.

The present invention encompasses methods of detecting, preventing,diagnosing, prognosticating, treating, and/or ameliorating a disease ordisorder. In preferred embodiments, the present invention encompasses amethod of treating diabetes mellitus comprising administering to apatient in which such detection, treatment, prevention, and/oramelioration is desired a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) in an amount effective todetect, prevent, diagnose, prognosticate, treat, and/or amelioratediabetes mellitus.

In another embodiment, the present invention also encompasses methods ofdetecting, preventing, diagnosing, prognosticating, treating, and/orameliorating diabetes mellitus;

comprising administering to a patient combinations of the proteins,nucleic acids, or antibodies of the invention (or fragments or variantsthereof), sharing similar indications as shown in the corresponding rowsin Column 3 of Table 1D.

Table 1D provides information related to biological activities forpolynucleotides and polypeptides of the invention (including antibodies,agonists, and/or antagonists thereof). Table 1D also providesinformation related to assays which may be used to test polynucleotidesand polypeptides of the invention (including antibodies, agonists,and/or antagonists thereof) for the corresponding biological activities.The first column (“Gene No.”) provides the gene number in theapplication for each clone identifier. The second column (“cDNA CloneID:”) provides the unique clone identifier for each clone as previouslydescribed and indicated in Table 1A through Table 1D. The third column(“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number forpolypeptide sequences encoded by the corresponding cDNA clones (also asindicated in Tables 1A, Table 1B, and Table 2). The fourth column(“Biological Activity”) indicates a biological activity corresponding tothe indicated polypeptides (or polynucleotides encoding saidpolypeptides). The fifth column (“Exemplary Activity Assay”) furtherdescribes the corresponding biological activity and also providesinformation pertaining to the various types of assays which may beperformed to test, demonstrate, or quantify the corresponding biologicalactivity.

Table 1D describes the use of, inter alia, FMAT technology for testingor demonstrating various biological activities. Fluorometric microvolumeassay technology (FMAT) is a fluorescence-based system which provides ameans to perform nonradioactive cell and bead-based assays to detectactivation of cell signal transduction pathways. This technology wasdesigned specifically for ligand binding and immunological assays. Usingthis technology, fluorescent cells or beads at the bottom of the wellare detected as localized areas of concentrated fluorescence using adata processing system. Unbound flurophore comprising the backgroundsignal is ignored, allowing for a wide variety of homogeneous assays.FMAT technology may be used for peptide ligand binding assays,immunofluorescence, apoptosis, cytotoxicity, and bead-basedimmunocapture assays. See, Miraglia S et. al., “Homogeneous cell andbead based assays for high throughput screening using flourometricmicrovolume assay technology,” Journal of Biomolecular Screening;4:193-204 (1999). In particular, FMAT technology may be used to test,confirm, and/or identify the ability of polypeptides (includingpolypeptide fragments and variants) to activate signal transductionpathways. For example, FMAT technology may be used to test, confirm,and/or identify the ability of polypeptides to up regulate production ofimmunomodulatory proteins (such as, for example, interleukins, GM-CSF,Rantes, and Tumor Necrosis factors, as well as other cellular regulators(e.g. insulin)).

Table 1D also describes the use of kinase assays for testing,demonstrating, or quantifying biological activity. In this regard, thephosphorylation and de-phosphorylation of specific amino acid residues(e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteinsprovides a fast, reversible means for activation and de-activation ofcellular signal transduction pathways. Moreover, cell signaltransduction via phosphorylation/de-phosphorylation is crucial to theregulation of a wide variety of cellular processes (e.g. proliferation,differentiation, migration, apoptosis, etc.). Accordingly, kinase assaysprovide a powerful tool useful for testing, confirming, and/oridentifying polypeptides (including polypeptide fragments and variants)that mediate cell signal transduction events via proteinphosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R.“Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998). TABLE 1D AASEQ Gene cDNA ID Biological No. Clone ID NO:Y Activity ExemplaryActivity Assay 1 H6EDM64 205 Insulin Secretion Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 2 H6EEU40 206 Regulation of Assays for the regulation of viabilityand proliferation of cells in vitro are well-known in the art viabilityand and may be used or routinely modified to assess the ability ofpolypeptides of the invention proliferation of (including antibodies andagonists or antagonists of the invention) to regulate viability andpancreatic beta proliferation of pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability cells. assay measures thenumber of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ohtani KI, etal., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Ex ClinEndocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 3 HACAB68 207 Activation of Assaysfor the activation of transcription through the cAMP response elementare well-known in transcription the art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughcAMP (including antibodies and agonists or antagonists of the invention)to increase cAMP and regulate response element CREB transcriptionfactors, and modulate expression of genes involved in a wide variety ofcell in immune cells functions. Exemplary assays for transcriptionthrough the cAMP response element that may be used (such as T-cells). orroutinely modified to test cAMP-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Black et al., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., JImmunol 161(2): 659-665 (1998), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse T cells that may be used according to theseassays include the CTLL cell line, which is a suspension culture of IL-2dependent cytotoxic T cells. 3 HACAB68 207 Activation of Assays for theactivation of transcription through the Serum Response Element (SRE) arewell- transcription known in the art and may be used or routinelymodified to assess the ability of polypeptides of the through seruminvention (including antibodies and agonists or antagonists of theinvention) to regulate the serum response element response factors andmodulate the expression of genes involved in growth. Exemplary assaysfor in immune cells transcription through the SRE that may be used orroutinely modified to test SRE activity of the (such as T-cells).polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension culture of T cells with cytotoxic activity. 3 HACAB68 207Activation of Kinase assay. JNK and p38 kinase assays for signaltransduction that regulate cell proliferation, Endothelial Cellactivation, or apoptosis are well known in the art and may be used orroutinely modified to assess p38 or JNK the ability of polypeptides ofthe invention (including antibodies and agonists or antagonists of theSignaling invention) to promote or inhibit cell proliferation,activation, and apoptosis. Exemplary assays for Pathway. JNK and p38kinase activity that may be used or routinely modified to test JNK andp38 kinase- induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Endothelial cells that may be used according to these assaysare publicly available (e.g., through the ATCC). Exemplary endothelialcells that may be used according to these assays include human umbilicalvein endothelial cells (HUVEC), which are endothelial cells which linevenous blood vessels, and are involved in functions that include, butare not limited to, angiogenesis, vascular permeability, vascular tone,and immune cell extravasation. 3 HACAB68 207 Activation of Kinase assay.Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signalSkeletal Mucle transduction that regulate glucose metabolism and cellsurvivial are well-known in the art and may Cell PI3 Kinase be used orroutinely modified to assess the ability of polypeptides of theinvention (including Signalling antibodies and agonists or antagonistsof the invention) to promote or inhibit glucose metabolism Pathway andcell survival. Exemplary assays for PI3 kinase activity that may be usedor routinely modified to test PI3 kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by reference inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 4 HACBJ56208 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art viability andand may be used or routinely modified to assess the ability ofpolypeptides of the invention proliferation of (including antibodies andagonists or antagonists of the invention) to regulate viability andpancreatic beta proliferation of pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability cells. viability assaymeasures the number of viable cells in culture based on quantitation ofthe ATP present which signals the presence of metabolically activecells. Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: FriedrichsenBN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al.,Endocrinology, 139(4): 1494-9 (1998); Hugl SR. et al., J Biol Chem 1998Jul 10; 273(28): 17771-9 (1998), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 5 HADMB15 209 Regulation of Assays for theregulation of transcription through the DMEF1 response element arewell-known in transcription via the art and may be used or routinelymodified to assess the ability of polypeptides of the invention DMEF1response (including antibodies and agonists or antagonists of theinvention) to activate the DMEF1 response element in element in areporter construct (such as that containing the GLUT4 promoter) and toregulate insulin adipocytes and production. The DMEF1 response elementis present in the GLUT4 promoter and binds to MEF2 pre-adipocytestranscription factor and another transcription factor that is requiredfor insulin regulation of Glut4 expression in skeletal muscle. GLUT4 isthe primary insulin-responsive glucose transporter in fat and muscletissue. Exemplary assays that may be used or routinely modified to testfor DMEF1 response element activity (in adipocytes and pre-adipocytes)by polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 5HADMB15 209 Regulation of Caspase Apoptosis. Assays for caspaseapoptosis are well known in the art and may be used or apoptosis ofroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and immune cells agonists or antagonistsof the invention) to regulate caspase protease-mediated apoptosis inimmune (such as mast cells (such as, for example, in mast cells). Mastcells are found in connective and mucosal tissues cells). throughout thebody, and their activation via immunoglobulin E-antigen, promoted by Thelper cell type 2 cytokines, is an important component of allergicdisease. Dysregulation of mast cell apoptosis may play a role inallergic disease and mast cell tumor survival. Exemplary assays forcaspase apoptosis that may be used or routinely modified to test capaseapoptosis activity induced by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Masuda A, et al., J Biol Chem, 276(28): 26107-26113(2001); Yeatman CF 2nd, et al., J Exp Med. 192(8): 1093-1103 (2000); Leeet al., FEBS Lett 485(2-3): 122- 126 (2000); Nor et al., J Vasc Res37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2):75-80 (1996); the contents of each of which are herein incorporated byreference in its entirety. Immune cells that may be used according tothese assays are publicly available (e.g., through commercial sources).Exemplary immune cells that may be used according to these assaysinclude mast cells such as the HMC human mast cell line. 5 HADMB15 209Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Natural Killer regulate cellproliferation or differentiation are well known in the art and may beused or routinely Cell ERK modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orSignaling antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Pathway. Exemplaryassays for ERK kinase activity that may be used or routinely modified totest ERK kinase-induced activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang andKarin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys MolBiol 71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Natural killer cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC). Exemplary natural killer cells that may be used according tothese assays include the human natural killer cell lines (for example,NK-YT cells which have cytolytic and cytotoxic activity) or primary NKcells. 6 HAGDW20 210 Upregulation of HLA-DR FMAT. MHC class II isessential for correct presentation of antigen to CD4+ T cells. HLA-DRand Deregulation of MHC class II has been associated with autoimmunediseases (e.g., diabetes, activation of rheumatoid arthritis, systemiclupus erythematosis, and multiple sclerosis). Assays for T cellsimmunomodulatory proteins expressed on MHC class II expressing T cellsand antigen presenting cells are well known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate the activation of T cells, and/or mediate humoralor cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of MHC class IIproducts, such as HLA-DR antigens, and the activation of T cells. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include, for example, theassays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6:138-160(2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992);Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher andZier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J HistochemCytochem 40(11): 1675-1683, the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or otherwise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 7 HAGEQ79 211 Activation of Assays for theactivation of transcription through the Gamma Interferon Activation Site(GAS) transcription response element are well-known in the art and maybe used or routinely modified to assess the through GAS ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the response element invention) to regulate STATtranscription factors and modulate gene expression involved in a wide inimmune cells variety of cell functions. Exemplary assays fortranscription through the GAS response element that (such as T-cells).may be used or routinely modified to test GAS-response element activityof polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al.,J Immunol 155(10): 4582-4587 (1995), the contents of each of which areherein incorporated by reference in its entirety. Exemplary mouse Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the CTLL cell line, which is a suspensionculture of IL-2 dependent cytotoxic T cells. 7 HAGEQ79 211 Upregulationof HLA-DR FMAT. MHC class II is essential for correct presentation ofantigen to CD4+ T cells. HLA-DR and Deregulation of MHC class II hasbeen associated with autoimmune diseases (e.g., diabetes, activation ofrheumatoid arthritis, systemic lupus erythematosis, and multiplesclerosis). Assays for T cells immunomodulatory proteins expressed onMHC class II expressing T cells and antigen presenting cells are wellknown in the art and may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate the activation ofT cells, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof MHC class II products, such as HLA-DR antigens, and the activation ofT cells. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222(1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itohet al., J Histochem Cytochem 40(11): 1675-1683, the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or otherwise known in the art. Human T cellsare primary human lymphocytes that mature in the thymus and express a TCell receptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be reactivated to enhance responsivenessto immunomodulatory factors. 8 HAJBV67 212 Stimulation of Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 9 HAJCH70 213 Activation of Kinase assay.Kinase assays, for example an GSK-3 assays, for PI3 kinase signaltransduction that Adipocyte PI3 regulate glucose metabolism and cellsurvival are well-known in the art and may be used or Kinase Signallingroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and Pathway agonists or antagonists ofthe invention) to promote or inhibit glucose metabolism and cellsurvival. Exemplary assays for PI3 kinase activity that may be used orroutinely modified to test PI3 kinase- induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is acontinous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 10 HAOAG15 214Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101- 1110(1998); Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2):126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Changand Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys MolBiol 71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 10 HAOAG15 214 Activationof Kinase assay. Kinase assays, for example an Elk-1 kinase assay, forERK signal transduction that Natural Killer regulate cell proliferationor differentiation are well known in the art and may be used orroutinely Cell ERK modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Signaling antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Pathway. Exemplary assays for ERK kinase activity thatmay be used or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101- 1110 (1998); Kyriakis JM, Biochem SocSymp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001);and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contentsof each of which are herein incorporated by reference in its entirety.Natural killer cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary natural killercells that may be used according to these assays include the humannatural killer cell lines (for example, NK-YT cells which have cytolyticand cytotoxic activity) or primary NK cells. 11 HATCD80 215 InsulinSecretion Assays for measuring secretion of insulin are well-known inthe art and may be used or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) to stimulate insulin secretion. Forexample, insulin secretion is measured by FMAT using anti-rat insulinantibodies. Insulin secretion from pancreatic beta cells is upregulatedby glucose and also by certain proteins/peptides, and disregulation is akey component in diabetes. Exemplary assays that may be used orroutinely modified to test for stimulation of insulin secretion (frompancreatic cells) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin: Shimizu, H., et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A.M., et al., Mol Endocrinol, 13(8): 1305-17 (1999); Filipsson, K., etal., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L. K., et al., J BiolChem, 271(28): 16544-52 (1996); and, Miraglia S et. al., Journal ofBiomolecular Screening, 4: 193-204 (1999), the contents of each of whichis herein incorporated by reference in its entirety. Pancreatic cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 12 HATCI03 216 Activation of Kinaseassay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinasesignal Skeletal Mucle transduction that regulate glucose metabolism andcell survivial are well-known in the art and may Cell PI3 Kinase be usedor routinely modified to assess the ability of polypeptides of theinvention (including Signalling antibodies and agonists or antagonistsof the invention) to promote or inhibit glucose metabolism Pathway andcell survival. Exemplary assays for PI3 kinase activity that may be usedor routinely modified to test PI3 kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by reference inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 12 HATCI03216 Upregulation of CD69 FMAT. CD69 is an activation marker that isexpressed on activated T cells, B cells, and NK CD69 and cells. CD69 isnot expressed on resting T cells, B cells, or NK cells. CD69 has beenfound to be activation of associated with inflammation. Assays forimmunomodulatory proteins expressed in T cells, B cells, T cells andleukocytes are well known in the art and may be used or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and agonists or antagonists of the invention) tomodulate the activation of T cells, and/or mediate humoral orcell-mediated immunity. Exemplary assays that test for immunomodulatoryproteins evaluate the upregulation of cell surface markers, such asCD69, and the activation of T cells. Such assays that may be used orroutinely modified to test immunomodulatory activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include, for example, the assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204 (1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Ferencziet al., J Autoimmun 14(1): 63-78 (200); Werfel et al., Allergy 52(4):465-469 (1997); Taylor-Fishwick and Siegel, Eur J Immunol 25(12):3215-3221 (1995); and Afetra et al., Ann Rheum Dis 52(6): 457-460(1993), the contents of each of which are herein incorporated byreference in its entirety. Human T cells that may be used according tothese assays may be isolated using techniques disclosed herein orotherwise known in the art. Human T cells are primary human lymphocytesthat mature in the thymus and express a T Cell receptor and CD3, CD4, orCD8. These cells mediate humoral or cell-mediated immunity and may bepreactivated to enhance responsiveness to immunomodulatory factors. 13HBAGD86 217 Regulation of Assays for the regulation of transcriptionthrough the DMEF1 response element are well-known in transcription viathe art and may be used or routinely modified to assess the ability ofpolypeptides of the invention DMEF1 response (including antibodies andagonists or antagonists of the invention) to activate the DMEF1 responseelement in element in a reporter construct (such as that containing theGLUT4 promoter) and to regulate insulin adipocytes and production. TheDMEF1 response element is present in the GLUT4 promoter and binds toMEF2 pre-adipocytes transcription factor and another transcriptionfactor that is required for insulin regulation of Glut4 expression inskeletal muscle. GLUT4 is the primary insulin-responsive glucosetransporter in fat and muscle tissue. Exemplary assays that may be usedor routinely modified to test for DMEF1 response element activity (inadipocytes and pre-adipocytes) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed inThai, M. V., et al., J Biol Chem, 273(23):14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21): 16323-8 (2000);Liu, M. L., et al., J Biol Chem, 269(45): 28514-21 (1994);“Identification of a 30-base pair regulatory element and novel DNAbinding protein that regulates the human GLUT4 promoter in transgenicmice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, et al., Gene66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216:362-368 (1992), the contents of each of which is herein incorporated byreference in its entirety. Adipocytes and pre-adipocytes that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary cells that may beused according to these assays include the mouse 3T3-L1 cell line whichis an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are acontinuous substrain of 3T3 fibroblasts developed through clonalisolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 13HBAGD86 217 Activation of Assays for the activation of transcriptionthrough the cAMP response element are well-known in the transcriptionart and may be used or routinely modified to assess the ability ofpolypeptides of the invention through cAMP (including antibodies andagonists or antagonists of the invention) to increase cAMP, regulateresponse element CREB transcription factors, and modulate expression ofgenes involved in a wide variety of cell (CRE) in pre- functions. Forexample, a 3T3-L1/CRE reporter assay may be used to identify factorsthat activate adipocytes. the cAMP signaling pathway. CREB plays a majorrole in adipogenesis, and is involved in differentiation intoadipocytes. CRE contains the binding sequence for the transcriptionfactor CREB (CRE binding protein). Exemplary assays for transcriptionthrough the cAMP response element that may be used or routinely modifiedto test cAMP-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923(1998), the contents of each of which are herein incorporated byreference in its entirety. Pre- adipocytes that may be used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 13 HBAGD86 217 Activationof Assays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate the serum response elementresponse factors and modulate the expression of genes involved ingrowth. Exemplary assays for in pre-adipocytes. transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety.Pre-adipocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary mouse adipocyte cells that may be used according to theseassays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocytecell line that is a continuous substrain of 3T3 fibroblast cellsdeveloped through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 13 HBAGD86 217 Activation of This reporter assaymeasures activation of the GATA-3 signaling pathway in HMC-1 human masttranscription cell line. Activation of GATA-3 in mast cells has beenlinked to cytokine and chemokine through GATA-3 production. Assays forthe activation of transcription through the GATA3 response element areresponse element well-known in the art and may be used or routinelymodified to assess the ability of polypeptides of in immune cells theinvention (including antibodies and agonists or antagonists of theinvention) to regulate GATA3 (such as mast transcription factors andmodulate expression of mast cell genes important for immune responsecells). development. Exemplary assays for transcription through theGATA3 response element that may be used or routinely modified to testGATA3-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring HarbSymp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur JImmunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596(1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), thecontents of each of which are herein incorpor- ated by reference in itsentirety. Mast cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary human mast cellsthat may be used acc- ording to these assays include the HMC-1 cellline, which is an immature human mast cell line established from theperipheral blood of a patient with mast cell leukemia, and exhibits manycharacteristics of immature mast cells. 13 HBAGD86 217 Activation ofThis reporter assay measures activation of the NFAT signaling pathway inHMC-1 human mast cell transcription line. Activation of NFAT in mastcells has been linked to cytokine and chemokine production. through NFATAssays for the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) response element response element arewell-known in the art and may be used or routinely modified to assessthe in immune cells ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the (such as mast invention)to regulate NFAT transcription factors and modulate expression of genesinvolved in cells). immunomodulatory functions. Exemplary assays fortranscription through the NFAT response element that may be used orroutinely modified to test NFAT-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali etal., J Immunol 165(12): 7215- 7223 (2000); Hutchinson and McCloskey, JBiol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188:527—537 (1998), the contents of each of which are herein incorporated byreference in its entirety. Mast cells that may be used according tothese assays are publicly available (e.g., through the ATCC). Exemplaryhuman mast cells that may be used according to these assays include theHMC-1 cell line, which is an immature human mast cell line establishedfrom the peripheral blood of a patient with mast cell leukemia, andexhibits many characteristics of immature mast cells. 13 HBAGD86 217Activation of This reporter assay measures activation of the NFkBsignaling pathway in HMC-1 human mast cell transcription line.Activation of NFkB in mast cells has been linked to production ofcertain cytokines, such as through NFKB IL-6 and IL-9. Assays for theactivation of transcription through the NFKB response element areresponse element well-known in the art and may be used or routinelymodified to assess the ability of polypeptides of in immune cells theinvention (including antibodies and agonists or antagonists of theinvention) to regulate NFKB (such as mast transcription factors andmodulate expression of immunomodulatory genes. Exemplary assays forcells). transcription through the NFKB response element that may be usedor rountinely modified to test NFKB-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Stassen et al, J Immunol 166(7): 4391-8 (2001); and Marquardt andWalker, J Allergy Clin Immunol 105(3): 500-5 (2000), the contents ofeach of which are herein incorporated by reference in its entirety. Mastcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary human mast cells that may be usedaccording to these assays include the HMC-1 cell line, which is animmature human mast cell line established from the peripheral blood of apatient with mast cell leukemia, and exhibits many characteristics ofimmature mast cells. 13 HBAGD86 217 Activation of This reporter assaymeasures activation of the NFkB signaling pathway in Ku812 humanbasophil transcription cell line. Assays for the activation oftranscription through the NFKB response element are well- through NFKBknown in the art and may be used or routinely modified to assess theability of polypeptides of the response element invention (includingantibodies and agonists or antagonists of the invention) to regulateNFKB in immune cells transcription factors and modulate expression ofimmunomodulatory genes. Exemplary assays for (such as transcriptionthrough the NFKB response element that may be used or rountinelymodified to test basophils). NFKB-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), thecontents of each of which are herein incorporated by reference in itsentirety. Basophils that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary human basophilcell lines that may be used according to these assays include Ku812,originally established from a patient with chronic myelogenous leukemia.It is an immature prebasophilic cell line that can be induced todifferentiate into mature basophils. 13 HBAGD86 217 Activation of Assaysfor the activation of transcription through the Serum Response Element(SRE) are well- transcription known in the art and may be used orroutinely modified to assess the ability of polypeptides of the throughserum invention (including antibodies and agonists or antagonists of theinvention) to bind the serum response element response factor andmodulate the expression of genes involved in growth and upregulate thein immune cells function of growth-related genes in many cell types.Exemplary assays for transcription through the (such as T-cells). SREthat may be used or routinely modified to test SRE activity of thepolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Benson et al., J Immunol 153(9): 3862-3873 (1994); and Black et al.,Virus Genes 12(2): 105-117 (1997), the content of each of which areherein incorporated by reference in its entirety. T cells that may beused according to these assays are publicly available (e.g., through theATCC). Exemplary human T cells, such as the MOLT4, that may be usedaccording to these assays are publicly available (e.g., through theATCC). 13 HBAGD86 217 Activation of Assays for the activation oftranscription through the Signal Transducers and Activators oftranscription Transcription (STAT6) response element are well-known inthe art and may be used or routinely through STAT6 modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or response element antagonists of the invention) to regulateSTAT6 transcription factors and modulate the expression of in immunecells multiple genes. Exemplary assays for transcription through theSTAT6 response element that may (such as natural be used or routinelymodified to test STAT6 response element activity of the polypeptides ofthe killer cells). invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al.,Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38):29331-29337 (2000), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary rat natural killer cells that may be used according tothese assays are publicly available (e.g., through the ATCC). 13 HBAGD86217 Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to regulate STAT transcription factors and modulategene expression involved in a wide in immune cells variety of cellfunctions. Exemplary assays for transcription through the GAS responseelement that (such as T-cells). may be used or routinely modified totest GAS-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6):1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587(1995), the contents of each of which are herein incorporated byreference in its entirety. Exemplary human T cells, such as the SUPTcell line, that may be used according to these assays are publiclyavailable (e.g., through the ATCC). 13 HBAGD86 217 Activation of Assaysfor the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) transcription response element are well-knownin the art and may be used or routinely modified to assess the throughNFAT ability of polypeptides of the invention (including antibodies andagonists or antagonists of the response element invention) to regulateNFAT transcription factors and modulate expression of genes involved inin immune cells immunomodulatory functions. Exemplary assays fortranscription through the NFAT response (such as natural element thatmay be used or routinely modified to test NFAT-response element activityof killer cells). polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser etal., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem268(19): 14285-14293 (1993), the contents of each of which are hereinincorporated by reference in its entirety. NK cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human NK cells that may be used according to theseassays include the NK-YT cell line, which is a human natural killer cellline with cytolytic and cytotoxic activity. 14 HBHAA05 218 Regulation ofAssays for the regulation of viability and proliferation of cells invitro are well-known in the art and viability and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 15 HBHAA81 219 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 4414 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 15 HBHAA81 219 Production of IFNgamma FMAT. IFNg plays a centralrole in the immune system and is considered to be a IFNgamma usingproinflammatory cytokine. IFNg promotes TH1 and inhibits TH2differentiation; promotes IgG2a a T cells and inhibits IgE secretion;induces macrophage activation; and increases MHC expression. Assays forimmunomodulatory proteins produced by T cells and NK cells that regulatea variety of inflammatory activities and inhibit TH2 helper cellfunctions are well known in the art and may be used or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and agonists or antagonists of the invention) tomediate immunomodulation, regulate inflammatory activities, modulate TH2helper cell function, and/or mediate humoral or cell- mediated immunity.Exemplary assays that test for immunomodulatory proteins evaluate theproduction of cytokines, such as Interferon gamma (IFNg), and theactivation of T cells. Such assays that may be used or routinelymodified to test immunomodulatory activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Miraglia et al., JBiomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes:a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J ClinLab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856:22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), andRheumatology (Oxford) 38(3): 214-20 (1999), the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or other- wise known in the art. Human Tcells are primary human lymphocytes that mature in the thymus andexpress a T Cell receptor and CD3, CD4, or CD8. These cells mediatehumoral or cell-mediated immunity and may be preactivated to enhanceresponsiveness to immunomodulatory factors. 16 HBIAA59 220 Activation ofKinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERKsignal transduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101- 1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 17 HBJAC40 221Regulation of Assays for the regulation of viability and proliferationof cells in vitro are well-known in the art and viability and may beused or routinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1):172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1):29-34 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 18 HBJCR46 222 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artand viability and may be used or routinely modified to assess theability of polypeptides of the invention (including proliferation ofantibodies and agonists or antagonists of the invention) to regulateviability and proliferation of pancreatic beta pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures the cells. number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely modified to test regulation of viability and proliferation ofpancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48(2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); HuglSR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), thecontents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include rat INS-1 cells. INS-1 cells are a semi-adherentcell line established from cells isolated from an X-ray induced rattransplantable insulinoma. These cells retain characteristics typical ofnative pancreatic beta cells including glucose inducible insulinsecretion. References: Asfari et al. Endocrinology 1992 130: 167. 19HBJDW56 223 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: FriedrichsenBN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al.,Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998Jul 10; 273(28): 17771-9 (1998), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 20 HBJEL16 224 Regulation of Assays for theregulation of viability and proliferation of cells in vitro arewell-known in the art and viability and may be used or routinelymodified to assess the ability of polypeptides of the invention(including proliferation of antibodies and agonists or antagonists ofthe invention) to regulate viability and proliferation of pancreaticbeta pancreatic beta cells. For example, the Cell Titer-Glo luminescentcell viability assay measures the cells. number of viable cells inculture based on quantitation of the ATP present which signals thepresence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 20 HBJEL16 224 Production of Assays formeasuring expression of VCAM are well-known in the art and may be usedor routinely VCAM in modified to assess the ability of polypeptides ofthe invention (including antibodies and agonists or endothelial cellsantagonists of the invention) to regulate VCAM expression. For example,FMAT may be used to (such as human meaure the upregulation of cellsurface VCAM-1 expresssion in endothelial cells. Endothelial cellsumbilical vein are cells that line blood vessels, and are involved infunctions that include, but are not limited to, endothelial cellsangiogenesis, vascular permeability, vascular tone, and immune cellextravasation. Exemplary (HUVEC)) endothelial cells that may be usedaccording to these assays include human umbilical vein endothelial cells(HUVEC), which are available from commercial sources. The expression ofVCAM (CD106), a membrane-associated protein, can be upregulated bycytokines or other factors, and contributes to the extravasation oflymphocytes, leucocytes and other immune cells from blood vessels; thusVCAM expression plays a role in promoting immune and inflammatoryresponses. 20 HBJEL16 224 Upregulation of CD69 FMAT. CD69 is anactivation marker that is expressed on activated T cells, B cells, andNK CD69 and cells. CD69 is not expressed on resting T cells, B cells, orNK cells. CD69 has been found to be activation of associated withinflammation. Assays for immunomodulatory proteins expressed in T cells,B cells, T cells and leukocytes are well known in the art and may beused or routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate the activation of T cells, and/or mediate humoralor cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of cell surfacemarkers, such as CD69, and the activation of T cells. Such assays thatmay be used or routinely modified to test immunomodulatory activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include, for example, the assays disclosedin Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowlandet al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Ferenczi et al., J Autoimmun 14(1): 63-78 (200); Werfel et al., Allergy52(4): 465-469 (1997); Taylor-Fishwick and Siegel, Eur J Immunol 25(12):3215-3221 (1995); and Afetra et al., Ann Rheum Dis 52(6): 457-460(1993), the contents of each of which are herein incorporated byreference in its entirety. Human T cells that may be used according tothese assays may be isolated using techniques disclosed herein orotherwise known in the art. Human T cells are primary human lymphocytesthat mature in the thymus and express a T Cell receptor and CD3, CD4, orCD8. These cells mediate humoral or cell-mediated immunity and may bepreactivated to enhance responsiveness to immunomodulatory factors. 21HBJIG20 225 Activation of Kinase assay. Kinase assays, for example anGSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transductionthat regulate glucose metabolism and cell survivial are well-known inthe art and may Cell PI3 Kinase be used or routinely modified to assessthe ability of polypeptides of the invention (including Signallingantibodies and agonists or antagonists of the invention) to promote orinhibit glucose metabolism Pathway and cell survival. Exemplary assaysfor PI3 kinase activity that may be used or routinely modified to testPI3 kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Forrer et al., Biol Chem 379(8- 9): 1101-1110 (1998);Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al.,Diabetes 48(8): 1662-1666 (1999), the contents of each of which areherein incorporated by reference in its entirety. Rat myoblast cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 22 HBJKD16 226 Production ofMIP-1alpha FMAT. Assays for immunomodulatory proteins produced byactivated dendritic cells MIP1alpha that upregulate monocyte/macrophageand T cell chemotaxis are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation, modulate chemotaxis, andmodulate T cell differentiation. Exemplary assays that test forimmunomodulatory proteins evaluate the production of chemokines, such asmacrophage inflammatory protein 1 alpha (MIP-1a), and the activation ofmonocytes/macrophages and T cells. Such assays that may be used orroutinely modified to test immunomodulatory and chemotaxis activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204(1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes etal., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828(1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 22 HBJKD16 226 Production of IL-6 FMAT. IL-6 isproduced by T cells and has strong effects on B cells. IL-6 participatesin IL-4 IL-6 induced IgE production and increases IgA production (IgAplays a role in mucosal immunity). IL-6 induces cytotoxic T cells.Deregulated expression of IL-6 has been linked to autoimmune disease,plasmacytomas, myelomas, and chronic hyperproliferative diseases. Assaysfor immunomodulatory and differentiation factor proteins produced by alarge variety of cells where the expression level is strongly regulatedby cytokines, growth factors, and hormones are well known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to mediate immunomodulation and differentiation andmodulate T cell proliferation and function. Exemplary assays that testfor immunomodulatory proteins evaluate the production of cytokines, suchas IL-6, and the stimulation and upregulation of T cell proliferationand functional activities. Such assays that may be used or routinelymodified to test immunomodulatory and dififerentiation activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204(1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); andVerhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of eachof which are herein incorporated by reference in its entirety. Humandendritic cells that may be used according to these assays may beisolated using techniques disclosed herein or otherwise known in theart. Human dendritic cells are antigen presenting cells in suspensionculture, which, when activated by antigen and/or cytokines, initiate andupregulate T cell proliferation and functional activities. 22 HBJKD16226 Stimulation of Assays for measuring calcium flux are well-known inthe art and may be used or routinely Calcium Flux in modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or pancreatic beta antagonists of the invention) to mobilizecalcium. For example, the FLPR assay may be used to cells. measureinflux of calcium. Cells normally have very low concentrations ofcytosolic calcium compared to much higher extracellular calcium.Extracellular factors can cause an influx of calcium, leading toactivation of calcium responsive signaling pathways and alterations incell functions. Exemplary assays that may be used or routinely modifiedto measure calcium flux by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995);Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB,et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al.,Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 23 HBMTY48 227 Activation of Kinaseassay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinasesignal Skeletal Mucle transduction that regulate glucose metabolism andcell survivial are well-known in the art and may Cell PI3 Kinase be usedor routinely modified to assess the ability of polypeptides of theinvention (including Signalling antibodies and agonists or antagonistsof the invention) to promote or inhibit glucose metabolism Pathway andcell survival. Exemplary assays for PI3 kinase activity that may be usedor routinely modified to test PI3 kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8- 9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by reference inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 24 HBMUH74228 Activation of Kinase assay. JNK kinase assays for signaltransduction that regulate cell proliferation, activation, JNK Signalingor apoptosis are well known in the art and may be used or routinelymodified to assess the ability of Pathway in polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to immune cells promote or inhibit cell proliferation,activation, and apoptosis. Exemplary assays for JNK kinase (such asactivity that may be used or routinely modified to test JNKkinase-induced activity of polypeptides eosinophils). of the invention(including antibodies and agonists or antagonists of the invention)include the assays disclosed in Forrer et al., Biol Chem 379(8-9):1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999);Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4):479-500 (1999); the contents of each of which are herein incorporated byreference in its entirety. Exemplary cells that may be used according tothese assays include eosinophils. Eosinophils are important in the latestage of allergic reactions; they are recruited to tissues and mediatethe inflammatory response of late stage allergic reaction. Moreover,exemplary assays that may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate signaltransduction, cell proliferation, activation, or apoptosis ineosinophils include assays disclosed and/or cited in: Zhang JP, et al.,“Role of caspases in dexamethasone-induced apoptosis and activation ofc-Jun NH2-terminal kinase and p38 mitogen- activated protein kinase inhuman eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7 (2000);Hebestreit H, et al., “Disruption of fas receptor signaling by nitricoxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); J AllergyClin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, et al., “Invivo resistance to corticosteroids in bronchial asthma is associatedwith enhanced phosyphorylation of JUN N-terminal kinase and failure ofprednisolone to inhibit JUN N-terminal kinase phosphorylation” J AllergyClin Immunol; Sep; 104(3 Pt 1): 565-74 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. 24 HBMUH74228 Regulation of Assays for the regulation of transcription of MalicEnzyme are well-known in the art and may be transcription of used orroutinely modified to assess the ability of polypeptides of theinvention (including Malic Enzyme in antibodies and agonists orantagonists of the invention) to regulate transcription of Malic Enzyme,a adipocytes key enzyme in lipogenesis. Malic enzyme is involved inlipogenesisand its expression is stimulted by insulin. ME promotercontains two direct repeat (DR1)- like elements MEp and MEd identifiedas putative PPAR response elements. ME promoter may also responds to AP1and other transcription factors. Exemplary assays that may be used orroutinely modified to test for regulation of transcription of MalicEnzyme (in adipoocytes) by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91(1998); Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9(1994); Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999);Ijpenberg, A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger,et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods inEnzymol. 216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Hepatocytes that may be usedaccording to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary hepatocytes that maybe used according to these assays includes the H4IIE rat liver hepatomacell line. 25 HBQAB79 229 Stimulation of Assays for measuring secretionof insulin are well-known in the art and may be used or routinelyinsulin secretion modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or from pancreaticantagonists of the invention) to stimulate insulin secretion. Forexample, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 26 HBXCX15 230 Regulation of Assays for theregulation of transcription through the DMEF1 response element arewell-known in transcription via the art and may be used or routinelymodified to assess the ability of polypeptides of the invention DMEF1response (including antibodies and agonists or antagonists of theinvention) to activate the DMEF1 response element in element in areporter construct (such as that containing the GLUT4 promoter) and toregulate insulin adipocytes and production. The DMEF1 response elementis present in the GLUT4 promoter and binds to MEF2 pre-adipocytestranscription factor and another transcription factor that is requiredfor insulin regulation of Glut4 expression in skeletal muscle. GLUT4 isthe primary insulin-responsive glucose transporter in fat and muscletissue. Exemplary assays that may be used or routinely modified to testfor DMEF1 response element activity (in adipocytes and pre-adipocytes)by polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 26HBXCX15 230 Activation of Assays for the activation of transcriptionthrough the cAMP response element are well-known in the transcriptionart and may be used or routinely modified to assess the ability ofpolypeptides of the invention through cAMP (including antibodies andagonists or antagonists of the invention) to increase cAMP, regulateresponse element CREB transcription factors, and modulate expression ofgenes involved in a wide variety of cell (CRE) in pre- functions. Forexample, a 3T3-L1/CRE reporter assay may be used to identify factorsthat activate adipocytes. the cAMP signaling pathway. CREB plays a majorrole in adipogenesis, and is involved in differentiation intoadipocytes. CRE contains the binding sequence for the transcriptionfactor CREB (CRE binding protein). Exemplary assays for transcriptionthrough the cAMP response element that may be used or routinely modifiedto test cAMP-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923(1998), the contents of each of which are herein incorporated byreference in its entirety. Pre- adipocytes that may be used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 26 HBXCX15 230 Activationof Assays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate the serum response elementresponse factors and modulate the expression of genes involved ingrowth. Exemplary assays for in pre-adipocytes. transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety.Pre-adipocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary mouse adipocyte cells that may be used according to theseassays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocytecell line that is a continuous substrain of 3T3 fibroblast cellsdeveloped through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 26 HBXCX15 230 Activation of This reporter assaymeasures activation of the GATA-3 signaling pathway in HMC-1 human masttranscription cell line. Activation of GATA-3 in mast cells has beenlinked to cytokine and chemokine through GATA-3 production. Assays forthe activation of transcription through the GATA3 response element areresponse element well-known in the art and may be used or routinelymodified to assess the ability of polypeptides of in immune cells theinvention (including antibodies and agonists or antagonists of theinvention) to regulate GATA3 (such as mast transcription factors andmodulate expression of mast cell genes important for immune responsecells). development. Exemplary assays for transcription through theGATA3 response element that may be used or routinely modified to testGATA3-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring HarbSymp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur JImmunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596(1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), thecontents of each of which are herein incorporated by reference in itsentirety. Mast cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary human mast cellsthat may be used according to these assays include the HMC-1 cell line,which is an immature human mast cell line established from theperipheral blood of a patient with mast cell leukemia, and exhibits manycharacteristics of immature mast cells. 26 HBXCX15 230 Activation ofThis reporter assay measures activation of the NFAT signaling pathway inHMC-1 human mast cell transcription line. Activation of NFAT in mastcells has been linked to cytokine and chemokine production. through NFATAssays for the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) response element response element arewell-known in the art and may be used or routinely modified to assessthe in immune cells ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the (such as mast invention)to regulate NFAT transcription factors and modulate expression of genesinvolved in cells). immunomodulatory functions. Exemplary assays fortranscription through the NFAT response element that may be used orroutinely modified to test NFAT-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali etal., J Immunol 165(12): 7215- 7223 (2000); Hutchinson and McCloskey, JBiol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188:527-537 (1998), the contents of each of which are herein incorporated byreference in its entirety. Mast cells that may be used according tothese assays are publicly available (e.g., through the ATCC). Exemplaryhuman mast cells that may be used according to these assays include theHMC-1 cell line, which is an immature human mast cell line establishedfrom the peripheral blood of a patient with mast cell leukemia, andexhibits many characteristics of immature mast cells. 26 HBXCX15 230Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to bind the serum responseelement response factor and modulate the expression of genes involved ingrowth and upregulate the in immune cells function of growth-relatedgenes in many cell types. Exemplary assays for transcription through the(such as T-cells). SRE that may be used or routinely modified to testSRE activity of the polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); andBlack et al., Virus Genes 12(2): 105-117 (1997), the content of each ofwhich are herein incorporated by reference in its entirety. T cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary human T cells, such as the MOLT4, that maybe used according to these assays are publicly available (e.g., throughthe ATCC). 26 HBXCX15 230 Activation of Assays for the activation oftranscription through the Signal Transducers and Activators oftranscription Transcription (STAT6) response element are well-known inthe art and may be used or routinely through STAT6 modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or response element antagonists of the invention) to regulateSTAT6 transcription factors and modulate the expression of in immunecells multiple genes. Exemplary assays for transcription through theSTAT6 response element that may (such as natural be used or routinelymodified to test STAT6 response element activity of the polypeptides ofthe killer cells). invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al.,Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38):29331-29337 (2000), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary rat natural killer cells that may be used according tothese assays are publicly available (e.g., through the ATCC). 26 HBXCX15230 Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to regulate STAT transcription factors and modulategene expression involved in a wide in immune cells variety of cellfunctions. Exemplary assays for transcription through the GAS responseelement that (such as T-cells). may be used or routinely modified totest GAS-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6):1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587(1995), the contents of each of which are herein incorporated byreference in its entirety. Exemplary human T cells, such as the SUPTcell line, that may be used according to these assays are publiclyavailable (e.g., through the ATCC). 26 HBXCX15 230 Activation of Assaysfor the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) transcription response element are well-knownin the art and may be used or routinely modified to assess the throughNFAT ability of polypeptides of the invention (including antibodies andagonists or antagonists of the response element invention) to regulateNFAT transcription factors and modulate expression of genes involved inin immune cells immunomodulatory functions. Exemplary assays fortranscription through the NFAT response (such as natural element thatmay be used or routinely modified to test NFAT-response element activityof killer cells). polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser etal., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem268(19): 14285-14293 (1993), the contents of each of which are hereinincorporated by reference in its entirety. NK cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human NK cells that may be used according to theseassays include the NK-YT cell line, which is a human natural killer cellline with cytolytic and cytotoxic activity. 26 HBXCX15 230 Activation ofAssays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate serum response elementresponse factors and modulate the expression of genes involved in growthand upregulate the in immune cells function of growth-related genes inmany cell types. Exemplary assays for transcription through the (such asnatural SRE that may be used or routinely modified to test SRE activityof the polypeptides of the invention killer cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873(1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the contentof each of which are herein incorporated by reference in its entirety. Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the NK-YT cell line, which is a human naturalkiller cell line with cytolytic and cytotoxic activity. 27 HCDCY76 231Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or Signaling routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and Pathway agonistsor antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 27 HCDCY76 231 EndothelialCell Caspase Apoptosis. Assays for caspase apoptosis are well known inthe art and may be used or Apoptosis routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to promote caspaseprotease-mediated apoptosis. Induction of apoptosis in endothelial cellssupporting the vasculature of tumors is associated with tumor regressiondue to loss of tumor blood supply. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Noret al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, JAtheroscler Thromb 3(2): 75-80 (1996); the contents of each of which areherein incorporated by reference in its entirety. Endothelial cells thatmay be used according to these assays are publicly available (e.g.,through commercial sources). Exemplary endothelial cells that may beused according to these assays include bovine aortic endothelial cells(bAEC), which are an example of endothelial cells which line bloodvessels and are involved in functions that include, but are not limitedto, angiogenesis, vascular permeability, vascular tone, and immune cellextravasation. 28 HCEDR26 232 Stimulation of Assays for measuringcalcium flux are well-known in the art and may be used or routinelyCalcium Flux in modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 29 HCEEU18 233 Activation of Kinase assay. Kinase assays, forexample an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 29 HCEEU18 233 Activationof Assays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate the serum response elementresponse factors and modulate the expression of genes involved ingrowth. Exemplary assays for in immune cells transcription through theSRE that may be used or routinely modified to test SRE activity of the(such as T-cells). polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117 (1997),the content of each of which are herein incorporated by reference in itsentirety. T cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary mouse T cellsthat may be used according to these assays include the CTLL cell line,which is an IL-2 dependent suspension culture of T cells with cytotoxicactivity. 30 HCEGX05 234 Activation of Kinase assay. Kinase assays, forexample an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 31 HCFLN88 235 Stimulationof Assays for measuring calcium flux are well-known in the art and maybe used or routinely Calcium Flux in modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orpancreatic beta antagonists of the invention) to mobilize calcium. Forexample, the FLPR assay may be used to cells. measure influx of calcium.Cells normally have very low concentrations of cytosolic calciumcompared to much higher extracellular calcium. Extracellular factors cancause an influx of calcium, leading to activation of calcium responsivesignaling pathways and alterations in cell functions. Exemplary assaysthat may be used or routinely modified to measure calcium flux bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Satin LS, etal., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al.,Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J,288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 31 HCFLN88 235 Activation of Assays for the activation oftranscription through the cAMP response element are well-known in thetranscription art and may be used or routinely modified to assess theability of polypeptides of the invention through cAMP (includingantibodies and agonists or antagonists of the invention) to increasecAMP, regulate response element CREB transcription factors, and modulateexpression of genes involved in a wide variety of cell in immune cellsfunctions. Exemplary assays for transcription through the cAMP responseelement that may be used (such as T-cells). or routinely modified totest cAMP-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2):105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998),the contents of each of which are herein incorporated by reference inits entirety. T cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary mouse T cellsthat may be used according to these assays include the HT2 cell line,which is a suspension culture of IL-2 dependent T cells that alsorespond to IL-4. 31 HCFLN88 235 Activation of Assays for the activationof transcription through the Serum Response Element (SRE) are well-transcription known in the art and may be used or routinely modified toassess the ability of polypeptides of the through serum invention(including antibodies and agonists or antagonists of the invention) toregulate serum response element response factors and modulate theexpression of genes involved in growth. Exemplary assays for in immunecells transcription through the SRE that may be used or routinelymodified to test SRE activity of the (such as T-cells). polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Benson etal., J Immunol 153(9): 3862-3873 (1994); and Black et al., Virus Genes12(2): 105-117 (1997), the content of each of which are hereinincorporated by reference in its entirety. Mouse T cells that may beused according to these assays are publicly available (e.g., through theATCC). Exemplary mouse T cells that may be used according to theseassays include the HT2 cell line, which is an IL-2 dependent suspensionculture of T cells that also respond to IL-4. 32 HCHAB84 236 Stimulationof Assays for measuring calcium flux are well-known in the art and maybe used or routinely Calcium Flux in modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orpancreatic beta antagonists of the invention) to mobilize calcium. Forexample, the FLPR assay may be used to cells. measure influx of calcium.Cells normally have very low concentrations of cytosolic calciumcompared to much higher extracellular calcium. Extracellular factors cancause an influx of calcium, leading to activation of calcium responsivesignaling pathways and alterations in cell functions. Exemplary assaysthat may be used or routinely modified to measure calcium flux bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Satin LS, etal., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al.,Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J,288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 33 HCMSX51 237 Regulation of Caspase Apoptosis. Assays for caspaseapoptosis are well known in the art and may be used or apoptosis inroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and pancreatic beta agonists orantagonists of the invention) to promote caspase protease-mediatedapoptosis. cells. Apoptosis in pancreatic beta is associated withinduction and progression of diabetes. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 34HCNCO11 238 Stimulation of Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely insulin secretionmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or from pancreatic antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is beta cells. measured by FMAT using anti-rat insulinantibodies. Insulin secretion from pancreatic beta cells is upregulatedby glucose and also by certain proteins/peptides, and disregulation is akey component in diabetes. Exemplary assays that may be used orroutinely modified to test for stimulation of insulin secretion (frompancreatic cells) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li,M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al.,FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal ofBiomolecular Screening, 4: 193-204 (1999), the contents of each of whichis herein incorporated by reference in its entirety. Pancreatic cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays include ratINS-1 cells. INS-1 cells are a semi-adherent cell line established fromcells isolated from an X-ray induced rat transplantable insulinoma.These cells retain characteristics typical of native pancreatic betacells including glucose inducible insulin secretion. References: Asfariet al. Endocrinology 1992 130: 167. 35 HCNSD29 239 Stimulation of Assaysfor measuring secretion of insulin are well-known in the art and may beused or routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 36 HCQBH72 240 Regulation of Assays for theregulation of viability and proliferation of cells in vitro arewell-known in the art and viability and may be used or routinelymodified to assess the ability of polypeptides of the invention(including proliferation of antibodies and agonists or antagonists ofthe invention) to regulate viability and proliferation of pancreaticbeta pancreatic beta cells. For example, the Cell Titer-Glo luminescentcell viability assay measures the cells. number of viable cells inculture based on quantitation of the ATP present which signals thepresence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that maybe used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 36 HCQBH72 240 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 37 HCQCC96 241 Activation of Kinase assay. Kinase assays, forexample an GSK-3 assays, for PI3 kinase signal transduction thatAdipocyte PI3 regulate glucose metabolism and cell survival arewell-known in the art and may be used or Kinase Signalling routinelymodified to assess the ability of polypeptides of the invention(including antibodies and Pathway agonists or antagonists of theinvention) to promote or inhibit glucose metabolism and cell survival.Exemplary assays for PI3 kinase activity that may be used or routinelymodified to test PI3 kinase- induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is acontinous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 37 HCQCC96 241Regulation of Assays for the regulation of transcription through thePEPCK promoter are well-known in the art transcription and may be usedor routinely modified to assess the ability of polypeptides of theinvention through the (including antibodies and agonists or antagonistsof the invention) to activate the PEPCK promoter PEPCK promoter in areporter construct and regulate liver gluconeogenesis. Exemplary assaysfor regulation of in hepatocytes transcription through the PEPCKpromoter that may be used or routinely modified to test for PEPCKpromoter activity (in hepatocytes) of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Lochhead et al., Diabetes 49(6):896-903 (2000); and Yeagley et al., J Biol Chem 275(23): 17814-17820(2000), the contents of each of which is herein incorporated byreference in its entirety. Hepatocyte cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary liver hepatoma cells that may beused according to these assays include H4lle cells, which contain atyrosine amino transferase that is inducible with glucocorticoids,insulin, or cAMP derivatives. 37 HCQCC96 241 Activation of Kinase assay.Kinase assays, for examplek Elk-1 kinase assays, for ERK signaltransduction that Skeletal Muscle regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelyCell ERK modified to assess the ability of polypeptides of the invention(including antibodies and agonists or Signalling antagonists of theinvention) to promote or inhibit cell proliferation, activation, anddifferentiation. Pathway Exemplary assays for ERK kinase activity thatmay be used or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Rat myoblast cells that may be used according to these assaysare publicly available (e.g., through the ATCC). Exemplary rat myoblastcells that may be used according to these assays include L6 cells. L6 isan adherent rat myoblast cell line, isolated from primary cultures ofrat thigh muscle, that fuses to form multinucleated myotubes andstriated fibers after culture in differentiation media. 38 HCQCJ56 242Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 39 HCUDD64 243 Productionof GM-CSF FMAT. GM-CSF is expressed by activated T cells, macrophages,endothelial cells, and GM-CSF fibroblasts. GM-CSF regulatesdifferentiation and proliferation of granulocytes- macrophageprogenitors and enhances antimicrobial activity in neutrophils,monocytes and macrophage. Additionally, GM-CSF plays an important rolein the differentiation of dendritic cells and monocytes, and increasesantigen presentation. GM-CSF is considered to be a proinflammatorycytokine. Assays for immunomodulatory proteins that promote theproduction of GM-CSF are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation and modulate the growth anddifferentiation of leukocytes. Exemplary assays that test forimmunomodulatory proteins evaluate the production of cytokines, such asGM-CSF, and the activation of T cells. Such assays that may be used orroutinely modified to test immunomodulatory activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Miraglia et al., JBiomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes:a practical approach” Chapter 6: 138-160 (2000); and Ye et al., J LeukocBiol (58(2): 225-233, the contents of each of which are hereinincorporated by reference in its entirety. Natural killer cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC) or may be isolated using techniques disclosed herein orotherwise known in the art. Natural killer (NK) cells are large granularlymphocytes that have cytotoxic activity but do bind antigen. NK cellsshow antibody-independent killing of tumor cells and also recognizeantibody bound on target cells, via NK Fc receptors, leading tocell-mediated cytotoxicity. 39 HCUDD64 243 Regulation of CaspaseApoptosis. Assays for caspase apoptosis are well known in the art andmay be used or apoptosis in routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and pancreatic betaagonists or antagonists of the invention) to promote caspaseprotease-mediated apoptosis. cells. Apoptosis in pancreatic beta isassociated with induction and progression of diabetes. Exemplary assaysfor caspase apoptosis that may be used or routinely modified to testcapase apoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 40HCWAE64 244 Regulation of Assays for the regulation of transcriptionthrough the DMEF1 response element are well-known in transcription theart and may be used or routinely modified to assess the ability ofpolypeptides of the invention via DMEF1 (including antibodies andagonists or antagonists of the invention) to activate the DMEF1 responseresponse element element in a reporter construct (such as thatcontaining the GLUT4 promoter) and to regulate insulin in adipocytes andproduction. The DMEF1 response element is present in the GLUT4 promoterand binds to MEF2 pre-adipocytes transcription factor and anothertranscription factor that is required for insulin regulation of Glut4expression in skeletal muscle. GLUT4 is the primary insulin-responsiveglucose transporter in fat and muscle tissue. Exemplary assays that maybe used or routinely modified to test for DMEF1 response elementactivity (in adipocytes and pre-adipocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed inThai, M. V., et al., J Biol Chem,273(23): 14285-92 (1998); Mora, S., et al., J Biol Chem, 275(21):16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45): 28514-21(1994); “Identification of a 30-base pair regulatory element and novelDNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 40HCWAE64 244 Activation of Assays for the activation of transcriptionthrough the cAMP response element are well-known in the transcriptionart and may be used or routinely modified to assess the ability ofpolypeptides of the invention through cAMP (including antibodies andagonists or antagonists of the invention) to increase cAMP, regulateresponse element CREB transcription factors, and modulate expression ofgenes involved in a wide variety of cell (CRE) in pre- functions. Forexample, a 3T3-L1/CRE reporter assay may be used to identify factorsthat activate adipocytes. the cAMP signaling pathway. CREB plays a majorrole in adipogenesis, and is involved in differentiation intoadipocytes. CRE contains the binding sequence for the transcriptionfactor CREB (CRE binding protein). Exemplary assays for transcriptionthrough the cAMP response element that may be used or routinely modifiedto test cAMP-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Reusch et al., Mol Cell Biol20(3): 1008-1020 (2000); and Klemm et al., J Biol Chem 273: 917-923(1998), the contents of each of which are herein incorporated byreference in its entirety. Pre- adipocytes that may be used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 40 HCWAE64 244 Activationof Assays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate the serum response elementresponse factors and modulate the expression of genes involved ingrowth. Exemplary assays for in pre-adipocytes. transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety.Pre-adipocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary mouse adipocyte cells that may be used according to theseassays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocytecell line that is a continuous substrain of 3T3 fibroblast cellsdeveloped through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 40 HCWAE64 244 Activation of This reporter assaymeasures activation of the GATA-3 signaling pathway in HMC-1 human masttranscription cell line. Activation of GATA-3 in mast cells has beenlinked to cytokine and chemokine through GATA-3 production. Assays forthe activation of transcription through the GATA3 response element areresponse element well-known in the art and may be used or routinelymodified to assess the ability of polypeptides of in immune cells theinvention (including antibodies and agonists or antagonists of theinvention) to regulate GATA3 (such as mast transcription factors andmodulate expression of mast cell genes important for immune responsecells). development. Exemplary assays for transcription through theGATA3 response element that may be used or routinely modified to testGATA3-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring HarbSymp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur JImmunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596(1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), thecontents of each of which are herein incorpor- ated by reference in itsentirety. Mast cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary human mast cellsthat may be used acc- ording to these assays include the HMC-1 cellline, which is an immature human mast cell line established from theperipheral blood of a patient with mast cell leukemia, and exhibits manycharacteristics of immature mast cells. 40 HCWAE64 244 Activation ofThis reporter assay measures activation of the NFAT signaling pathway inHMC-1 human mast cell transcription line. Activation of NFAT in mastcells has been linked to cytokine and chemokine production. through NFATAssays for the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) response element response element arewell-known in the art and may be used or routinely modified to assessthe in immune cells ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the (such as mast invention)to regulate NFAT transcription factors and modulate expression of genesinvolved in cells). immunomodulatory functions. Exemplary assays fortranscription through the NFAT response element that may be used orroutinely modified to test NFAT-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Ali etal., J Immunol 165(12): 7215-7223 (2000); Hutchinson and McCloskey, JBiol Chem 270(27): 16333-16338 (1995), and Turner et al., J Exp Med 188:527-537 (1998), the contents of each of which are herein incorporated byreference in its entirety. Mast cells that may be used according tothese assays are publicly available (e.g., through the ATCC). Exemplaryhuman mast cells that may be used according to these assays include theHMC-1 cell line, which is an immature human mast cell line establishedfrom the peripheral blood of a patient with mast cell leukemia, andexhibits many characteristics of immature mast cells. 40 HCWAE64 244Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to bind the serum responseelement response factor and modulate the expression of genes involved ingrowth and upregulate the in immune cells function of growth-relatedgenes in many cell types. Exemplary assays for transcription through the(such as T-cells). SRE that may be used or routinely modified to testSRE activity of the polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873 (1994); andBlack et al., Virus Genes 12(2): 105-117 (1997), the content of each ofwhich are herein incorporated by reference in its entirety. T cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary human T cells, such as the MOLT4, that maybe used according to these assays are publicly available (e.g., throughthe ATCC). 40 HCWAE64 244 Activation of Assays for the activation oftranscription through the Signal Transducers and Activators oftranscription Transcription (STAT6) response element are well-known inthe art and may be used or routinely through STAT6 modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or response element antagonists of the invention) to regulateSTAT6 transcription factors and modulate the expression of in immunecells multiple genes. Exemplary assays for transcription through theSTAT6 response element that may (such as natural be used or routinelymodified to test STAT6 response element activity of the polypeptides ofthe killer cells). invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al.,Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38):29331-29337 (2000), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary rat natural killer cells that may be used according tothese assays are publicly available (e.g., through the ATCC). 40 HCWAE64244 Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to regulate STAT transcription factors and modulategene expression involved in a wide in immune cells variety of cellfunctions. Exemplary assays for transcription through the GAS responseelement that (such as T-cells). may be used or routinely modified totest GAS-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362- 368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6):1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587(1995), the contents of each of which are herein incorporated byreference in its entirety. Exemplary human T cells, such as the SUPTcell line, that may be used according to these assays are publiclyavailable (e.g., through the ATCC). 40 HCWAE64 244 Activation of Assaysfor the activation of transcription through the Nuclear Factor ofActivated T cells (NFAT) transcription response element are well-knownin the art and may be used or routinely modified to assess the throughNFAT ability of polypeptides of the invention (including antibodies andagonists or antagonists of the response element invention) to regulateNFAT transcription factors and modulate expression of genes involved inin immune cells immunomodulatory functions. Exemplary assays fortranscription through the NFAT response (such as natural element thatmay be used or routinely modified to test NFAT-response element activityof killer cells). polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods inEnzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Aramburu et al., J Exp Med 182(3): 801-810 (1995); DeBoer et al., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser etal., Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem268(19): 14285-14293 (1993), the contents of each of which are hereinincorporated by reference in its entirety. NK cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human NK cells that may be used according to theseassays include the NK-YT cell line, which is a human natural killer cellline with cytolytic and cytotoxic activity. 40 HCWAE64 244 Activation ofAssays for the activation of transcription through the Serum ResponseElement (SRE) are well- transcription known in the art and may be usedor routinely modified to assess the ability of polypeptides of thethrough serum invention (including antibodies and agonists orantagonists of the invention) to regulate serum response elementresponse factors and modulate the expression of genes involved in growthand upregulate the in immune cells function of growth-related genes inmany cell types. Exemplary assays for transcription through the (such asnatural SRE that may be used or routinely modified to test SRE activityof the polypeptides of the invention killer cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873(1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the contentof each of which are herein incorporated by reference in its entirety. Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the NK-YT cell line, which is a human naturalkiller cell line with cytolytic and cytotoxic activity. 41 HDPDJ58 245Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 41 HDPDJ58 245 Upregulationof CD71 FMAT. CD71 is the transferrin receptor. Transferrin is a majoriron carrying protein that is CD71 and essential for cell proliferation.CD71 is expressed predominantly on cells that are actively activation ofproliferating. Assays for immunomodulatory proteins expressed onactivated T cells, B cells, and T cells most proliferating cells arewell known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate the activation ofT cells, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof cell surface markers, such as CD71, and the activation of T cells.Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); and Afetra et al., Ann Rheum Dis52(6): 457-460 (1993), the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or otherwise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness toimmunomodulatory factors. 42 HDPFU43 246 Production of IL-6 FMAT. IL-6is produced by T cells and has strong effects on B cells. IL-6participates in IL-4 IL-6 induced IgE production and increases IgAproduction (IgA plays a role in mucosal immunity). IL-6 inducescytotoxic T cells. Deregulated expression of IL-6 has been linked toautoimmune disease, plasmacytomas, myelomas, and chronichyperproliferative diseases. Assays for immunomodulatory anddifferentiation factor proteins produced by a large variety of cellswhere the expression level is strongly regulated by cytokines, growthfactors, and hormones are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation and differentiation and modulateT cell proliferation and function. Exemplary assays that test forimmunomodulatory proteins evaluate the production of cytokines, such asIL-6, and the stimulation and upregulation of T cell proliferation andfunctional activities. Such assays that may be used or routinelymodified to test immunomodulatory and differentiation activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204(1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); andVerhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of eachof which are herein incorporated by reference in its entirety. Humandendritic cells that may be used according to these assays may beisolated using techniques disclosed herein or otherwise known in theart. Human dendritic cells are antigen presenting cells in suspensionculture, which, when activated by antigen and/or cytokines, initiate andupregulate T cell proliferation and functional activities. 42 HDPFU43246 Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to modulate gene expression (commonly via STATtranscription factors) involved in a in immune cells wide variety ofcell functions. Exemplary assays for transcription through the GASresponse (such as element that may be used or routinely modified to testGAS-response element activity of eosinophils). polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainenet al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol155(10): 4582-4587 (1995); the contents of each of which are hereinincorporated by reference in its entirety. Moreover, exemplary assaysthat may be used or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) to activate or inhibit activation ofimmune cells include assays disclosed and/or cited in: Mayumi M.,“EoL-1, a human eosinophilic cell line” Leuk Lymphoma; Jun; 7(3): 243-50(1992); Bhattacharya S, “Granulocyte macrophage colony-stimulatingfactor and interleukin-5 activate STAT5 and induce CIS1 mRNA in humanperipheral blood eosinophils” Am J Respir Cell Mol Biol; Mar; 24(3):312-6 (2001); and, Du J, et al., “Engagement of the CrkL adapter ininterleukin-5 signaling in eosinophils” J Biol Chem; Oct 20; 275(42):33167-75 (2000); the contents of each of which are herein incorporatedby reference in its entirety. Exemplary cells that may be used accordingto these assays include eosinophils. Eosinophils are a type of immunecell important in the late stage of allergic reactions; they arerecruited to tissues and mediate the inflammtory response of late stageallergic reaction. Increases in GAS mediated transcription ineosinophils is typically a result of STAT activation, normally a directconsequence of interleukin or other cytokine receptor stimulation (e.g.IL3, IL5 or GMCSF). 42 HDPFU43 246 Activation of Kinase assay. Kinaseassays, for example an GSK-3 kinase assay, for PI3 kinase signaltransduction Skeletal Mucle that regulate glucose metabolism and cellsurvivial are well-known in the art and may be used or Cell PI3 Kinaseroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and Signalling agonists or antagonistsof the invention) to promote or inhibit glucose metabolism and cellsurvival. Pathway Exemplary assays for PI3 kinase activity that may beused or routinely modified to test PI3 kinase- induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by reference inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 43 HDPGE24247 Insulin Secretion Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to stimulate insulinsecretion. For example, insulin secretion is measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9(2000); Salapatek, A.M., et al., Mol Endocrinol, 13(8):1305-17 (1999);Filipsson, K., et al., Ann NY Acad Sci, 865:441-4 (1998); Olson, L.K.,et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al.,Journal of Biomolecular Screening, 4:193-204 (1999), the contents ofeach of which is herein incorporated by reference in its entirety.Pancreatic cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary pancreatic cells that may be used according to these assaysinclude HITT15 Cells. HITT15 are an adherent epithelial cell lineestablished from Syrian hamster islet cells transformed with 5V40. Thesecells express glucagon, somatostatin, and glucocorticoid receptors. Thecells secrete insulin, which is stimulated by glucose and glucagon andsuppressed by soniatostatin or glucocorticoids. ATTC#CRL-1777 Refs: Lordand Ashcroft. Biochem. 1. 219: 547-551; Santerre et al. Proc. NatI.Acad. Sci. USA 78: 4339-4343, 1981. 44 HDPIU94 248 Regulation of Assaysfor the regulation of transcription of Malic Enzyme are well-known inthe art and may be transcription of used or routinely modified to assessthe ability of polypeptides of the invention (including Malic Enzyme inantibodies and agonists or antagonists of the invention) to regulatetranscription of Malic Enzyme, a hepatocytes key enzyme in lipogenesis.Malic enzyme is involved in lipogenesisand its expression is stimultedby insulin. ME promoter contains two direct repeat (DR1)- like elementsMEp and MEd identified as putative PPAR response elements. ME promotermay also responds to AP1 and other transcription factors. Exemplaryassays that may be used or routinely modified to test for regulation oftranscription of Malic Enzyme (in hepatocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Streeper, R. S., et al., MolEndocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., MolEndocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem,274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32):20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Hepatocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assaysincludes the mouse 3T3-L1 cell line. 3T3- L1 is a mouse preadipocytecell line (adherent). It is a continuous substrain of 3T3 fibroblastsdeveloped through clonal isolation. Cells undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation cultureconditions. 44 HDPIU94 248 Activation of Assays for the activation oftranscription through the NFKB response element are well-known in thetranscription art and may be used or routinely modified to assess theability of polypeptides of the invention through NFKB (includingantibodies and agonists or antagonists of the invention) to regulateNFKB transcription response element factors and modulate expression ofimmunomodulatory genes. Exemplary assays for transcription in immunecells through the NFKB response element that may be used or rountinelymodified to test NFKB- (such as EOL1 response element activity ofpolypeptides of the invention (including antibodies and agonists orcells). antagonists of the invention) include assays disclosed in Bergeret al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216:362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346(1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997);Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al.,29(3): 838-844 (1999), the contents of each of which are hereinincorporated by reference in its entirety. For example, a reporter assay(which measures increases in transcription inducible from a NFkBresponsive element in EOL-1 cells) may link the NFKB element to arepeorter gene and binds to the NFKB transcription factor, which isupregulated by cytokines and other factors. Exemplary immune cells thatmay be used according to these assays include eosinophils such as thehuman EOL-1 cell line of eosinophils. Eosinophils are a type of immunecell important in the allergic responses; they are recruited to tissuesand mediate the inflammtory response of late stage allergic reaction.Eol-1 is a human eosinophil cell line. 44 HDPIU94 248 Activation ofKinase assay. Kinase assays, for example an Elk-1 kinase assay, for ERKsignal transduction that Hepatocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); andCobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents ofeach of which are herein incorporated by reference in its entirety. Ratliver hepatoma cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary rat liverhepatoma cells that may be used according to these assays include H4llecells, which are known to respond to glucocorticoids, insulin, or cAMPderivatives. 44 HDPIU94 248 Regulation of Kinase assays, for example anElk-1 kinase assay for ERK signal transduction that regulates cellproliferation and/ proliferation or differentiation, are well known inthe art and may be used or routinely modified to or differentiationassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists in immune cells of the invention)to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary (such as mast assays for ERK kinase activitythat may be used or routinely modified to test ERK kinase-inducedcells). activity of polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include the assaysdisclosed in: Ali H, et al., J Immunol, 165(12): 7215-7223 (2000); TamSY, et al., Blood, 90(5): 1807-1820 (1997); Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Berra et al., Biochem Pharmacol 60(8):1171-1178 (2000); Gupta et al., Exp Cell Res 247(2): 495-504 (1999);Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, ProgBiophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of whichare herein incorporated by reference in its entirety. Exemplary immunecells that may be used according to these assays include human mastcells such as the HMC-1 cell line. 44 HDPIU94 248 Production of IFNgammaFMAT. IFNg plays a central role in the immune system and is consideredto be a IFNgamma using proinflammatory cytokine. IFNg promotes TH1 andinhibits TH2 differentiation; promotes IgG2a a T cells and inhibits IgEsecretion; induces macrophage activation; and increases MHC expression.Assays for immunomodulatory proteins produced by T cells and NK cellsthat regulate a variety of inflammatory activities and inhibit TH2helper cell functions are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation, regulate inflammatoryactivities, modulate TH2 helper cell function, and/or mediate humoral orcell- mediated immunity. Exemplary assays that test for immunomodulatoryproteins evaluate the production of cytokines, such as Interferon gamma(IFNg), and the activation of T cells. Such assays that may be used orroutinely modified to test immunomodulatory activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Miraglia et al., JBiomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes:a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J ClinLab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856:22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), andRheumatology (Oxford) 38(3): 214-20 (1999), the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or otherwise known in the art. Human T cellsare primary human lymphocytes that mature in the thymus and express a TCell receptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 45 HDPIY31 249 Regulation of Assays for theregulation of viability and proliferation of cells in vitro arewell-known in the art and viability and may be used or routinelymodified to assess the ability of polypeptides of the invention(including proliferation of antibodies and agonists or antagonists ofthe invention) to regulate viability and proliferation of pancreaticbeta pancreatic beta cells. For example, the Cell Titer-Gb luminescentcell viability assay measures the cells. number of viable cells inculture based on quantitation of the ATP present which signals thepresence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ohtani K., et al., Endocrinology, 139(1):172-8 (1998); Krautheim A, et al, Exp Chin Endocrinol Diabetes, 107 (1):29-34 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITTiS Cells. HJTT 15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV4O. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC#CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219:547-55 1; Santerre et al. Proc. Nail. Acad. Sci. USA 78: 4339-4343,1981. 46 HDPOC24 250 Production of Assay that measures the production ofthe chemokine interleukin-8 (IL-8) from immune cells (such IL-8 byimmune as the EOL-1 human eosinophil cell line) are well known in theart (for example, measurement of cells (such as the IL-8 production byFMAT) and may be used or routinely modified to assess the ability ofhuman EOL-1 polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to eosinophil cells) promoteor inhibit. Eosinophils are a type of immune cell important in allergicresponses; they are recruited to tissues and mediate the inflammtoryresponse of late stage allergic reaction. IL8 is a strongimmunomodulator and may have a potential proinflammatory role inimmunological diseases and disorders (such as allergy and asthma). 46HDPOC24 250 Insulin Secretion Assays for measuring secretion of insulinare well-known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to stimulateinsulin secretion. For example, insulin secretion is measured by FMATusing anti-rat insulin antibodies. Insulin secretion from pancreaticbeta cells is upregulated by glucose and also by certainproteins/peptides, and disregulation is a key component in diabetes.Exemplary assays that may be used or routinely modified to test forstimulation of insulin secretion (from pancreatic cells) by polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in: Shimizu, H., et al., EndocrJ, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8):1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4(1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996);and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 46 HDPOC24 250 Regulation of Caspase Apoptosis. Assays for caspaseapoptosis are well known in the art and may be used or apoptosis inroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and pancreatic beta agonists orantagonists of the invention) to promote caspase protease-mediatedapoptosis. cells. Apoptosis in pancreatic beta is associated withinduction and progression of diabetes. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 47HDPPD93 251 Activation of Kinase assay. Kinase assays, for example anGSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3regulate glucose metabolism and cell survival are well-known in the artand may be used or Kinase Signalling routinely modified to assess theability of polypeptides of the invention (including antibodies andPathway agonists or antagonists of the invention) to promote or inhibitglucose metabolism and cell survival. Exemplary assays for PI3 kinaseactivity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina etal., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes48(8): 1662-1666 (1999), the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3- L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 47 HDPPD93 251 Activationof Assays for the activation of transcription through the AP1 responseelement are known in the art and transcription may be used or routinelymodified to assess the ability of polypeptides of the invention(including through AP1 antibodies and agonists or antagonists of theinvention) to modulate growth and other cell functions. response elementExemplary assays for transcription through the AP1 response element thatmay be used or routinely in immune cells modified to test AP1-responseelement activity of polypeptides of the invention (including (such asT-cells). antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1988); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem272(49): 30806-30811 (1997); Chang et al., Mol Cell Biol 18(9):4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3): 838-844(1999), the contents of each of which are herein incorporated byreference in its entirety. Mouse T cells that may be used according tothese assays are publicly available (e.g., through the ATCC). Exemplarymouse T cells that may be used according to these assays include the HT2cell line, which is an IL-2 dependent suspension culture cell line thatalso responds to IL-4. 47 HDPPD93 251 Activation of Assays for theactivation of transcription through the AP1 response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughAP1 (including antibodies and agonists or antagonists of the invention)to modulate growth and other cell response element functions. Exemplaryassays for transcription through the AP1 response element that may beused in immune cells or routinely modified to test AP1-response elementactivity of polypeptides of the invention (such as T-cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49):30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998);and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents ofeach of which are herein incorporated by reference in its entirety.Human T cells that may be used according to these assays are publiclyavailable (e.g.. through the ATCC). Exemplary human T cells that may beused acc- ording to these assays include the SUPT cell line, which is anIL-2 and IL-4 responsive sus- pension-culture cell line. 47 HDPPD93 251Activation of Assays for the activation of transcription through theCD28 response element are well-known in the transcription art and may beused or routinely modified to assess the ability of polypeptides of theinvention through CD28 (including antibodies and agonists or antagonistsof the invention) to stimulate IL-2 expression in T response elementcells. Exemplary assays for transcription through the CD28 responseelement that may be used or in immune cells routinely modified to testCD28-response element activity of polypeptides of the invention (such asT-cells). (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); McGuireand Iacobelli, J Immunol 159(3): 1319-1327 (1997); Parra et al., JImmunol 166(4): 2437-2443 (2001); and Butscher et al., J Biol Chem 3(1):552-560 (1998), the contents of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary humanT cells that may be used according to these assays include the SUPT cellline, which is a suspension culture of IL-2 and IL-4 responsive T cells.47 HDPPD93 251 Activation of Assays for the activation of transcriptionthrough the Nuclear Factor of Activated T cells (NFAT) transcriptionresponse element are well-known in the art and may be used or routinelymodified to assess the through NFAT ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theresponse element invention) to regulate NFAT transcription factors andmodulate expression of genes involved in in immune cellsimmunomodulatory functions. Exemplary assays for transcription throughthe NFAT response (such as T-cells). element that may be used orroutinely modified to test NFAT-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Serfling et al., Biochim Biophys Acta 1498(1): 1-18 (2000); De Boer etal., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al.,Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem268(19): 14285-14293 (1993), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human T cells that may be used according to theseassays include the SUPT cell line, which is a suspension culture of IL-2and IL-4 responsive T cells. 47 HDPPD93 251 Activation of Assays for theactivation of transcription through the NFKB response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughNFKB (including antibodies and agonists or antagonists of the invention)to regulate NFKB transcription response element factors and modulateexpression of immunomodulatory genes. Exemplary assays for transcriptionin immune cells through the NFKB response element that may be used orrountinely modified to test NFKB- (such as T-cells). response elementactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Black et al., Virus Gnes 15(2): 105-117 (1997); andFraser et al., 29(3): 838-844 (1999), the contents of each of which areherein incorporated by reference in its entirety. T cells that may beused according to these assays are publicly available (e.g., through theATCC). Exemplary human T cells that may be used according to theseassays include the SUPT cell line, which is a suspension culture of IL-2and IL-4 responsive T cells. 47 HDPPD93 251 Production of Assays forproduction of IL-10 and activation of T-cells are well known in the artand may be used IL-10 and or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and activation ofagonists or antagonists of the invention) to stimulate or inhibitproduction of IL-10 and/or activation T-cells. of T-cells. Exemplaryassays that may be used or routinely modified to assess the ability ofpolypeptides and antibodies of the invention (including agonists orantagonists of the invention) to modulate IL-10 production and/or T-cellproliferation include, for example, assays such as disclosed and/orcited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” BrMed Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2cell-directed therapy for asthma” Pharmacology & Therapeutics; 88:187-196 (2000); the contents of each of which are herein incorporated byreference in their entirety. Exemplary cells that may be used accordingto these assays include Th2 cells. IL10 secreted from Th2 cells may bemeasured as a marker of Th2 cell activation. Th2 cells are a class of Tcells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that inducedifferentiation and activation of Th2 cells play a major role in theinitiation and pathogenesis of allergy and asthma. Primary T helper 2cells are generated via in vitro culture under Th2 polarizing conditionsusing peripheral blood lymphocytes isolated from cord blood. 47 HDPPD93251 Activation of Assays for the activation of transcription through theNuclear Factor of Activated T cells (NFAT) transcription responseelement are well-known in the art and may be used or routinely modifiedto assess the through NFAT ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to regulate NFAT transcription factors and modulateexpression of genes involved in in immune cells immunomodulatoryfunctions. Exemplary assays for transcription through the NFAT response(such as natural element that may be used or routinely modified to testNFAT-response element activity of killer cells). polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburuet al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J BiochemCell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3):838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293(1993), the contents of each of which are herein incorporated byreference in its entirety. NK cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary humanNK cells that may be used according to these assays include the NK-YTcell line, which is a human natural killer cell line with cytolytic andcytotoxic activity. 48 HDPPQ30 252 Production of Endothelial cells,which are cells that line blood vessels, and are involved in functionsthat include, ICAM in but are not limited to, angiogenesis, vascularpermeability, vascular tone, and immune cell endothelial cellsextravasation. Exemplary endothelial cells that may be used in ICAMproduction assays include (such as human human umbilical veinendothelial cells (HUVEC), and are available from commercial sources.The umbilical vein expression of ICAM (CD54), a intergral membraneprotein, can be upregulated by cytokines or other endothelial cellsfactors, and ICAM expression is important in mediating immune andendothelial cell interactions (HUVEC)) leading to immune andinflammatory responses. Assays for measuring expression of ICAM-1 arewell-known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to regulate ICAM-1 expression.Exemplary assays that may be used or routinely modified to measureICAM-1 expression include assays disclosed in: Rolfe BE, et al.,Atherosclerosis, 149(1): 99-110 (2000); Panettieri RA Jr, et al., JImmunol, 154(5): 2358-2365 (1995); and, Grunstein MM, et al., Am JPhysiol Lung Cell Mol Physiol, 278(6): L1154-L1163 (2000), the contentsof each of which is herein incorporated by reference in its entirety. 48HDPPQ30 252 Stimulation of Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely insulin secretionmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or from pancreatic antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is beta cells. measured by FMAT using anti-rat insulinantibodies. Insulin secretion from pancreatic beta cells is upregulatedby glucose and also by certain proteins/peptides, and disregulation is akey component in diabetes. Exemplary assays that may be used orroutinely modified to test for stimulation of insulin secretion (frompancreatic cells) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li,M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al.,FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal ofBiomolecular Screening, 4: 193-204 (1999), the contents of each of whichis herein incorporated by reference in its entirety. Pancreatic cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays include ratINS-1 cells. INS-1 cells are a semi-adherent cell line established fromcells isolated from an X-ray induced rat transplantable insulinoma.These cells retain characteristics typical of native pancreatic betacells including glucose inducible insulin secretion. References: Asfariet al. Endocrinology 1992 130: 167. 49 HDQHM36 253 Insulin SecretionAssays for measuring secretion of insulin are well-known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann NY AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 50 HDTFX18 254 Activation of Kinase assay. Kinase assays, forexample an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 51 HE2CM39 255 Regulationof Assays for the regulation of transcription through the DMEF1 responseelement are well-known in transcription via the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention DMEF1 response (including antibodies and agonists orantagonists of the invention) to activate the DMEF1 response element inelement in a reporter construct (such as that containing the GLUT4promoter) and to regulate insulin adipocytes and production. The DMEF1response element is present in the GLUT4 promoter and binds to MEF2pre-adipocytes transcription factor and another transcription factorthat is required for insulin regulation of Glut4 expression in skeletalmuscle. GLUT4 is the primary insulin-responsive glucose transporter infat and muscle tissue. Exemplary assays that may be used or routinelymodified to test for DMEF1 response element activity (in adipocytes andpre-adipocytes) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Thai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S.,et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J BiolChem, 269(45): 28514-21 (1994); “Identification of a 30-base pairregulatory element and novel DNA binding protein that regulates thehuman GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4;275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Adipocytes and pre-adipocytes that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary cells that may be used according to these assaysinclude the mouse 3T3-L1 cell line which is an adherent mousepreadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of3T3 fibroblasts developed through clonal isolation. These cells undergoa pre-adipocyte to adipose-like conversion under appropriatedifferentiation culture conditions. 52 HE2HC60 256 Activation of Kinaseassay. Kinase assays, for example an GSK-3 kinase assay, for PI3 kinasesignal Skeletal Mucle transduction that regulate glucose metabolism andcell survivial are well-known in the art and may Cell PI3 Kinase be usedor routinely modified to assess the ability of polypeptides of theinvention (including Signalling antibodies and agonists or antagonistsof the invention) to promote or inhibit glucose metabolism Pathway andcell survival. Exemplary assays for PI3 kinase activity that may be usedor routinely modified to test PI3 kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by ref- erence inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 53 HE2PO93257 Activation of Kinase assay. Kinase assays, for example an GSK-3assays, for P13 kinase signal transduction that Adipocyte PI3 regulateglucose metabolism and cell survival are well-known in the art and maybe used or Kinase Signalling routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and Pathway agonistsor antagonists of the invention) to promote or inhibit glucosemetabolism and cell survival. Exemplary assays for P13 kinase activitythat may be used or routinely modified to test P13 kinase- inducedactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inForrer et al., Biol Chem 379(8-9):110l-1110 (1998); Nikoulina et al.,Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8):1662-1666 (1999), the contents of each of which are herein incorporatedby reference in its entirety. Mouse adipocyte cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse adipocyte cells that may be used according tothese assays include 3T3- Li cells. 3T3-L1 is an adherent mousepreadipocyte cell line that is a continous substrain of 3T3 fibroblastcells developed through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 53 HE2PO93 257 Activation of Assays for the activationof transcription through the Serum Response Element (SRE) are well-transcription known in the art and may be used or routinely modified toassess the ability of polypeptides of the through serum invention(including antibodies and agonists or antagonists of the invention) toregulate the serum response element response factors and modulate theexpression of genes involved in growth. Exemplary assays for in immunecells transcription through the SRE that may be used or routinelymodified to test SRE activity of the (such as T-cells). polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Blacket al., Virus Genes 12(2): 105-117 (1997), the content of each of whichare herein incorporated by reference in its entirety. T cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC). Exemplary mouse T cells that may be used according to theseassays include the CTLL cell line, which is an IL-2 dependent suspensionculture of T cells with cytotoxic activity. 54 HE6FU11 258 Regulation ofCaspase Apoptosis. Assays for caspase apoptosis are well known in theart and may be used or apoptosis in routinely modified to assess theability of polypeptides of the invention (including antibodies andpancreatic beta agonists or antagonists of the invention) to promotecaspase protease-mediated apoptosis. cells. Apoptosis in pancreatic betais associated with induction and progression of diabetes. Exemplaryassays for caspase apoptosis that may be used or routinely modified totest capase apoptosis activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include the assays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3):285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36(1996); Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000);Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., JImmunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2):238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Leeet al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2):75-80 (1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: # CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 55HE6FV29 259 Endothelial Cell Caspase Apoptosis. Assays for caspaseapoptosis are well known in the art and may be used or Apoptosisroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to promote caspase protease-mediated apoptosis. Induction ofapoptosis in endothelial cells supporting the vasculature of tumors isassociated with tumor regression due to loss of tumor blood supply.Exemplary assays for caspase apoptosis that may be used or routinelymodified to test capase apoptosis activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Lee et al., FEBS Lett485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000);and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); thecontents of each of which are herein incorporated by reference in itsentirety. Endothelial cells that may be used according to these assaysare publicly available (e.g., through commercial sources). Exemplaryendothelial cells that may be used according to these assays includebovine aortic endothelial cells (bAEC), which are an example ofendothelial cells which line blood vessels and are involved in functionsthat include, but are not limited to, angiogenesis, vascularpermeability, vascular tone, and immune cell extravasation. 55 HE6FV29259 Stimulation of Assays for measuring calcium flux are well-known inthe art and may be used or routinely Calcium Flux in modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or pancreatic beta antagonists of the invention) to mobilizecalcium. For example, the FLPR assay may be used to cells. measureinflux of calcium. Cells normally have very low concentrations ofcytosolic calcium compared to much higher extracellular calcium.Extracellular factors can cause an influx of calcium, leading toactivation of calcium responsive signaling pathways and alterations incell functions. Exemplary assays that may be used or routinely modifiedto measure calcium flux by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995);Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB,et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al.,Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 56 HE8TY46 260 Activation of Kinaseassay. Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Hepatocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101- 1110(1998); Kyriakis JM, Biochem Soc Symp64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); andCobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents ofeach of which are herein incorporated by reference in its entirety. Ratliver hepatoma cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary rat liverhepatoma cells that may be used according to these assays include H4llecells, which are known to respond to glucocorticoids, insulin, or cAMPderivatives. 57 HE9EA10 261 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artand viability and may be used or routinely modified to assess theability of polypeptides of the invention (including proliferation ofantibodies and agonists or antagonists of the invention) to regulateviability and proliferation of pancreatic beta pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures the cells. number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely modified to test regulation of viability and proliferation ofpancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48(2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); HuglSR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), thecontents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include rat INS-1 cells. INS-1 cells are a semi-adherentcell line established from cells isolated from an X-ray induced rattransplantable insulinoma. These cells retain characteristics typical ofnative pancreatic beta cells including glucose inducible insulinsecretion. References: Asfari et al. Endocrinology 1992 130: 167. 58HEBCY54 262 Regulation of Assays for the regulation of transcriptionthrough the FAS promoter element are well-known in the transcription artand may be used or routinely modified to assess the ability ofpolypeptides of the invention through the FAS (including antibodies andagonists or antagonists of the invention) to activate the FAS promoterpromoter element element in a reporter construct and to regulatetranscription of FAS, a key enzyme for lipogenesis. in hepatocytes FASpromoter is regulated by many transcription factors including SREBP.Insulin increases FAS gene transcription in livers of diabetic mice.This stimulation of transcription is also somewhat glucose dependent.Exemplary assays that may be used or routinely modified to test for FASpromoter element activity (in hepatocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Xiong, S., et al., Proc Natl AcadSci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem,260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65(1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al.,Methods in Enzymol. 216: 362-368 (1992), the contents of each of whichis herein incorporated by reference in its entirety. Hepatocytes thatmay be used according to these assays, such as H4IIE cells, are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assays includerat liver hepatoma cell line(s) inducible with glucocorticoids, insulin,or cAMP derivatives. 59 HEBFR46 263 Activation of Assays for theactivation of transcription through the cAMP response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughcAMP (including antibodies and agonists or antagonists of the invention)to increase cAMP, regulate response element CREB transcription factors,and modulate expression of genes involved in a wide variety of cell(CRE) in pre- functions. For example, a 3T3-L1/CRE reporter assay may beused to identify factors that activate adipocytes. the cAMP signalingpathway. CREB plays a major role in adipogenesis, and is involved indifferentiation into adipocytes. CRE contains the binding sequence forthe transcription factor CREB (CRE binding protein). Exemplary assaysfor transcription through the cAMP response element that may be used orroutinely modified to test cAMP-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000); and Klemm et al.,J Biol Chem 273: 917-923 (1998), the contents of each of which areherein incorporated by reference in its entirety. Pre- adipocytes thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 59 HEBFR46 263Activation of This reporter assay measures activation of the GATA-3signaling pathway in HMC-1 human mast transcription cell line.Activation of GATA-3 in mast cells has been linked to cytokine andchemokine through GATA-3 production. Assays for the activation oftranscription through the GATA3 response element are response elementwell-known in the art and may be used or routinely modified to assessthe ability of polypeptides of in immune cells the invention (includingantibodies and agonists or antagonists of the invention) to regulateGATA3 (such as mast transcription factors and modulate expression ofmast cell genes important for immune response cells). development.Exemplary assays for transcription through the GATA3 response elementthat may be used or routinely modified to test GATA3-response elementactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64:563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12):3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); andHenderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents ofeach of which are herein incorporated by reference in its entirety. Mastcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary human mast cells that may be usedaccording to these assays include the HMC-1 cell line, which is animmature human mast cell line established from the peripheral blood of apatient with mast cell leukemia, and exhibits many characteristics ofimmature mast cells. 59 HEBFR46 263 Stimulation of Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 59 HEBFR46 263 Activation of Assays for theactivation of transcription through the AP1 response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughAP1 (including antibodies and agonists or antagonists of the invention)to modulate growth and other cell response element functions. Exemplaryassays for transcription through the AP1 response element that may beused in immune cells or routinely modified to test AP1-response elementactivity of polypeptides of the invention (such as T-cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49):30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998);and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents ofeach of which are herein incorporated by reference in its entirety.Human T cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary human T cells that may beused according to these assays include the SUPT cell line, which is anIL-2 and IL-4 responsive suspension-culture cell line. 59 HEBFR46 263Activation of Assays for the activation of transcription through theCD28 response element are well-known in the transcription art and may beused or routinely modified to assess the ability of polypeptides of theinvention through CD28 (including antibodies and agonists or antagonistsof the invention) to stimulate IL-2 expression in T response elementcells. Exemplary assays for transcription through the CD28 responseelement that may be used or in immune cells routinely modified to testCD28-response element activity of polypeptides of the invention (such asT-cells). (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); McGuireand Iacobelli, J Immunol 159(3): 1319-1327 (1997); Parra et al., JImmunol 166(4): 2437-2443 (2001); and Butscher et al., J Biol Chem 3(1):552-560 (1998), the contents of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary humanT cells that may be used according to these assays include the SUPT cellline, which is a suspension culture of IL-2 and IL-4 responsive T cells.59 HEBFR46 263 Activation of Assays for the activation of transcriptionthrough the Nuclear Factor of Activated T cells (NFAT) transcriptionresponse element are well-known in the art and may be used or routinelymodified to assess the through NFAT ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theresponse element invention) to regulate NFAT transcription factors andmodulate expression of genes involved in in immune cellsimmunomodulatory functions. Exemplary assays for transcription throughthe NFAT response (such as T-cells). element that may be used orroutinely modified to test NFAT-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Serfling et al., Biochim Biophys Acta 1498(1): 1-18 (2000); De Boer etal., Int J Biochem Cell Biol 31(10): 1221-1236 (1999); Fraser et al.,Eur J Immunol 29(3): 838-844 (1999); and Yeseen et al., J Biol Chem268(19): 14285-14293 (1993), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human T cells that may be used according to theseassays include the SUPT cell line, which is a suspension culture of IL-2and IL-4 responsive T cells. 59 HEBFR46 263 Activation of Assays for theactivation of transcription through the Signal Transducers andActivators of transcription Transcription (STAT6) response element arewell-known in the art and may be used or routinely through STAT6modified to assess the ability of polypeptides of the invention(including antibodies and agonists or response element antagonists ofthe invention) to regulate STAT6 transcription factors and modulate theexpression of in immune cells multiple genes. Exemplary assays fortranscription through the STAT6 response element that may (such asT-cells). be used or routinely modified to test STAT6 response elementactivity of the polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362- 368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffattet al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur JImmunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38):29331-29337 (2000), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary T cells that may be used according to these assaysinclude the SUPT cell line, which is a suspension culture of IL-2 andIL-4 responsive T cells. 59 HEBFR46 263 Activation of Assays for theactivation of transcription through the NFKB response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughNFKB (including antibodies and agonists or antagonists of the invention)to regulate NFKB transcription response element factors and modulateexpression of immunomodulatory genes. Exemplary assays for transcriptionin immune cells through the NFKB response element that may be used orrountinely modified to test NFKB- (such as T-cells). response elementactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Black et al., Virus Gnes 15(2): 105-117 (1997); andFraser et al., 29(3): 838-844 (1999), the contents of each of which areherein incorporated by reference in its entirety. T cells that may beused according to these assays are publicly available (e.g., through theATCC). Exemplary human T cells that may be used according to theseassays include the SUPT cell line, which is a suspension culture of IL-2and IL-4 responsive T cells. 60 HEBGE07 264 Activation of Assays for theactivation of transcription through the Gamma Interferon Activation Site(GAS) transcription response element are well-known in the art and maybe used or routinely modified to assess the through GAS ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the response element invention) to regulate STATtranscription factors and modulate gene expression involved in a wide inimmune cells variety of cell functions. Exemplary assays fortranscription through the GAS response element that (such as T-cells).may be used or routinely modified to test GAS-response element activityof polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al.,J Immunol 155(10): 4582-4587 (1995), the contents of each of which areherein incorporated by reference in its entirety. Exemplary mouse Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the CTLL cell line, which is a suspensionculture of IL-2 dependent cytotoxic T cells. 60 HEBGE07 264 Activationof This reporter assay measures activation of the GATA-3 signalingpathway in HMC-1 human mast transcription cell line. Activation ofGATA-3 in mast cells has been linked to cytokine and chemokine throughGATA-3 production. Assays for the activation of transcription throughthe GATA3 response element are response element well-known in the artand may be used or routinely modified to assess the ability ofpolypeptides of in immune cells the invention (including antibodies andagonists or antagonists of the invention) to regulate GATA3 (such asmast transcription factors and modulate expression of mast cell genesimportant for immune response cells). development. Exemplary assays fortranscription through the GATA3 response element that may be used orroutinely modified to test GATA3-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362- 368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999);Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zhengand Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol CellBiol 14(6): 4286-4294 (1994), the contents of each of which are hereinincor- porated by reference in its entirety. Mast cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human mast cells that may be used according to theseassays include the HMC-1 cell line, which is an immature human mast cellline established from the peripheral blood of a patient with mast cellleukemia, and exhibits many characteristics of immature mast cells. 60HEBGE07 264 Stimulation of Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely insulin secretionmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or from pancreatic antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is beta cells. measured by FMAT using anti-rat insulinantibodies. Insulin secretion from pancreatic beta cells is upregulatedby glucose and also by certain proteins/peptides, and disregulation is akey component in diabetes. Exemplary assays that may be used orroutinely modified to test for stimulation of insulin secretion (frompancreatic cells) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li,M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al.,FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal ofBiomolecular Screening, 4: 193-204 (1999), the contents of each of whichis herein incorporated by reference in its entirety. Pancreatic cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays include ratINS-1 cells. INS-1 cells are a semi-adherent cell line established fromcells isolated from an X-ray induced rat transplantable insulinoma.These cells retain characteristics typical of native pancreatic betacells including glucose inducible insulin secretion. References: Asfariet al. Endocrinology 1992 130: 167. 61 HEGAU15 265 Stimulation of Assaysfor measuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 ( Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 62 HETCI16 266 Activation of Kinase assay. Kinase assays, forexample an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 63 HFCEI04 267 Regulationof Assays for the regulation of transcription through the DMEF1 responseelement are well-known in transcription via the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention DMEF1 response (including antibodies and agonists orantagonists of the invention) to activate the DMEF1 response element inelement in a reporter construct (such as that containing the GLUT4promoter) and to regulate insulin adipocytes and production. The DMEF1response element is present in the GLUT4 promoter and binds to MEF2pre-adipocytes transcription factor and another transcription factorthat is required for insulin regulation of Glut4 expression in skeletalmuscle. GLUT4 is the primary insulin-responsive glucose transporter infat and muscle tissue. Exemplary assays that may be used or routinelymodified to test for DMEF1 response element activity (in adipocytes andpre-adipocytes) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedinThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S.,et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J BiolChem, 269(45): 28514-21 (1994); “Identification of a 30-base pairregulatory element and novel DNA binding protein that regulates thehuman GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4;275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Adipocytes and pre-adipocytes that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary cells that may be used according to these assaysinclude the mouse 3T3-L1 cell line which is an adherent mousepreadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of3T3 fibroblasts developed through clonal isolation. These cells undergoa pre-adipocyte to adipose-like conversion under appropriatedifferentiation culture conditions. 64 HFCFE20 268 Activation of Kinaseassay. Kinase assays, for example an GSK-3 assays, for PI3 kinase signaltransduction that Adipocyte PI3 regulate glucose metabolism and cellsurvival are well-known in the art and may be used or Kinase Signallingroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and Pathway agonists or antagonists ofthe invention) to promote or inhibit glucose metabolism and cellsurvival. Exemplary assays for PI3 kinase activity that may be used orroutinely modified to test PI3 kinase- induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 65 HFPCZ55 269Regulation of Assays for the regulation of viability and proliferationof cells in vitro are well-known in the art and viability and may beused or routinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR. et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 66 HFPDR62 270 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 67 HFPDS07 271Activation of Assays for the activation of transcription through thecAMP response element are well-known in the transcription art and may beused or routinely modified to assess the ability of polypeptides of theinvention through cAMP (including antibodies and agonists or antagonistsof the invention) to increase cAMP, regulate response element CREBtranscription factors, and modulate expression of genes involved in awide variety of cell (CRE) in pre- functions. For example, a 3T3-L1/CREreporter assay may be used to identify factors that activate adipocytes.the cAMP signaling pathway. CREB plays a major role in adipogenesis, andis involved in differentiation into adipocytes. CRE contains the bindingsequence for the transcription factor CREB (CRE binding protein).Exemplary assays for transcription through the cAMP response elementthat may be used or routinely modified to test cAMP-response elementactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Reusch et al., Mol Cell Biol 20(3): 1008-1020 (2000);and Klemm et al., J Biol Chem 273: 917-923 (1998), the contents of eachof which are herein incorporated by reference in its entirety. Pre-adipocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary mouse adipocyte cells that may be used according to theseassays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocytecell line that is a continuous substrain of 3T3 fibroblast cellsdeveloped through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 67 HFPDS07 271 Activation of Kinase assay. Kinaseassays, for example an Elk-1 kinase assay, for ERK signal transductionthat Natural Killer regulate cell proliferation or differentiation arewell known in the art and may be used or routinely Cell ERK modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Signaling antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Pathway. Exemplary assays for ERK kinase activity that may be used orroutinely modified to test ERK kinase-induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Naturalkiller cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary natural killer cells thatmay be used according to these assays include the human natural killercell lines (for example, NK-YT cells which have cytolytic and cytotoxicactivity) or primary NK cells. 67 HFPDS07 271 Upregulation of HLA-DRFMAT. MHC class II is essential for correct presentation of antigen toCD4+ T cells. HLA-DR and Deregulation of MHC class II has beenassociated with autoimmune diseases (e.g., diabetes, activation ofrheumatoid arthritis, systemic lupus erythematosis, and multiplesclerosis). Assays for T cells immunomodulatory proteins expressed onMHC class II expressing T cells and antigen presenting cells are wellknown in the art and may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate the activation ofT cells, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof MHC class II products, such as HLA-DR antigens, and the activation ofT cells. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222(1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itohet al., J Histochem Cytochem 40(11): 1675-1683, the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or otherwise known in the art. Human T cellsare primary human lymphocytes that mature in the thymus and express a TCell receptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 68 HFVHW43 272 Stimulation of Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 69 HFXAV37 273 Activation of Kinase assay.Kinase assays, for example an GSK-3 kinase assay, for PI3 kinase signalSkeletal Mucle transduction that regulate glucose metabolism and cellsurvivial are well-known in the art and may Cell PI3 Kinase be used orroutinely modified to assess the ability of polypeptides of theinvention (including Signalling antibodies and agonists or antagonistsof the invention) to promote or inhibit glucose metabolism Pathway andcell survival. Exemplary assays for PI3 kinase activity that may be usedor routinely modified to test PI3 kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Forrer et al.,Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2):263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999),the contents of each of which are herein incorporated by ref- erence inits entirety. Rat myoblast cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary ratmyoblast cells that may be used according to these assays include L6cells. L6 is an adherent rat myoblast cell line, isolated from primarycultures of rat thigh muscle, that fuses to form multinucleated myotubesand striated fibers after culture in differentiation media. 70 HFXFZ46274 Upregulation of HLA-DR FMAT. MHC class II is essential for correctpresentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHCclass II has been associated with autoimmune diseases (e.g., diabetes,activation of rheumatoid arthritis, systemic lupus erythematosis, andmultiple sclerosis). Assays for T cells immunomodulatory proteinsexpressed on MHC class II expressing T cells and antigen presentingcells are well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to modulate theactivation of T cells, and/or mediate humoral or cell-mediated immunity.Exemplary assays that test for immunomodulatory proteins evaluate theupregulation of MHC class II products, such as HLA-DR antigens, and theactivation of T cells. Such assays that may be used or routinelymodified to test immunomodulatory activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include, for example, the assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204 (1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour etal., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, ImmunolLett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1):22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683,the contents of each of which are herein incorporated by reference inits entirety. Human T cells that may be used according to these assaysmay be isolated using techniques disclosed herein or otherwise known inthe art. Human T cells are primary human lymphocytes that mature in thethymus and express a T Cell receptor and CD3, CD4, or CD8. These cellsmediate humoral or cell- mediated immunity and may be preactivated toenhance responsiveness to immunomodulatory factors. 71 HGBHP91 275Regulation of Assays for the regulation of transcription through theDMEF1 response element are well-known in transcription via the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention DMEF1 response (including antibodies and agonists orantagonists of the invention) to activate the DMEF1 response element inelement in a reporter construct (such as that containing the GLUT4promoter) and to regulate insulin adipocytes and production. The DMEF1response element is present in the GLUT4 promoter and binds to MEF2pre-adipocytes transcription factor and another transcription factorthat is required for insulin regulation of Glut4 expression in skeletalmuscle. GLUT4 is the primary insulin-responsive glucose transporter infat and muscle tissue. Exemplary assays that may be used or routinelymodified to test for DMEF1 response element activity (in adipocytes andpre-adipocytes) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedinThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S.,et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J BiolChem, 269(45): 28514-21 (1994); “Identification of a 30-base pairregulatory element and novel DNA binding protein that regulates thehuman GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4;275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Adipocytes and pre-adipocytes that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary cells that may be used according to these assaysinclude the mouse 3T3-L1 cell line which is an adherent mousepreadipocyte cell line. Mouse 3T3-L1 cells are a continuous. substrainof 3T3 fibroblasts developed through clonal isolation. These cellsundergo a pre-adipocyte to adipose-like conversion under appropriatedifferentiation culture conditions. 72 HHEAK45 276 Insulin SecretionAssays for measuring secretion of insulin are well-known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 73 HHEOW19 277 Regulation of Assays for the regulation (i.e.increases or decreases) of viability and proliferation of cells in vitroviability or are well-known in the art and may be used or routinelymodified to assess the ability of polypeptides proliferation of of theinvention (including antibodies and agonists or antagonists of theinvention) to regulate immune cells viability and proliferation ofeosinophil cells and cell lines. For example, the CellTiter-Gloô (suchas human Luminescent Cell Viability Assay (Promega Corp., Madison, WI,USA ) can be used to measure the eosinophil EOL-1 number of viable cellsin culture based on quantitation of the ATP present which signals thecells). presence of metabolically active cells. Eosinophils are a typeof immune cell important in allergic responses; they are recruited totissues and mediate the inflammtory response of late stage allergicreaction. Eosinophil cell lines that may be used according to theseassays are publicly available and/or may be routinely generated.Exemplary eosinophil cells that may be used according to these assaysinclude EOL-1 Cells. 73 HHEOW19 277 Production of TNFa FMAT. Assays forimmunomodulatory proteins produced by activated macrophages, T cells,TNF alpha by fibroblasts, smooth muscle, and other cell types that exerta wide variety of inflammatory and dendritic cells cytotoxic effects ona variety of cells are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation, modulate inflammation andcytotoxicity. Exemplary assays that test for immunomodulatory proteinsevaluate the production of cytokines such as tumor necrosis factor alpha(TNFa), and the induction or inhibition of an inflammatory or cytotoxicresponse. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999);Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198);Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., JImmunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65:822-828 (1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 73 HHEOW19 277 Production of MIP-1alpha FMAT.Assays for immunomodulatory proteins produced by activated dendriticcells MIP1alpha that upregulate monocyte/macrophage and T cellchemotaxis are well known in the art and may be used or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and agonists or antagonists of the invention) tomediate immunomodulation, modulate chemotaxis, and modulate T celldifferentiation. Exemplary assays that test for immunomodulatoryproteins evaluate the production of chemokines, such as macrophageinflammatory protein 1 alpha (MIP-1a), and the activation ofmonocytes/macrophages and T cells. Such assays that may be used orroutinely modified to test immunomodulatory and chemotaxis activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204(1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); Drakes etal., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., J Immunol158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828(1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 73 HHEOW19 277 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 74 HHFFS40 278 Endothelial Cell Caspase Apoptosis. Assays forcaspase apoptosis are well known in the art and may be used or Apoptosisroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to promote caspase protease-mediated apoptosis. Induction ofapoptosis in endothelial cells supporting the vasculature of tumors isassociated with tumor regression due to loss of tumor blood supply.Exemplary assays for caspase apoptosis that may be used or routinelymodified to test capase apoptosis activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Lee et al., FEBS Lett485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000);and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); thecontents of each of which are herein incorporated by reference in itsentirety. Endothelial cells that may be used according to these assaysare publicly available (e.g., through commercial sources). Exemplaryendothelial cells that may be used according to these assays includebovine aortic endothelial cells (bAEC), which are an example ofendothelial cells which line blood vessels and are involved in functionsthat include, but are not limited to, angiogenesis, vascularpermeability, vascular tone, and immune cell extravasation. 74 HHFFS40278 Production of TNFa FMAT. Assays for immunomodulatory proteinsproduced by activated macrophages, T cells, TNF alpha by fibroblasts,smooth muscle, and other cell types that exert a wide variety ofinflammatory and dendritic cells cytotoxic effects on a variety of cellsare well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to mediateimmunomodulation, modulate inflammation and cytotoxicity. Exemplaryassays that test for immunomodulatory proteins evaluate the productionof cytokines such as tumor necrosis factor alpha (TNFa), and theinduction or inhibition of an inflammatory or cytotoxic response. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inMiraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland etal., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen etal., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828(1999), the contents of each of which are herein incorporated by ref-erence in its entirety. Human dendritic cells that may be used accordingto these assays may be iso- lated using techniques disclosed herein orotherwise known in the art. Human dendritic cells are antigen presentingcells in suspension culture, which, when activated by antigen and/orcytokines, initiate and upregulate T cell proliferation and functionalactivities. 74 HHFFS40 278 Stimulation of Assays for measuring calciumflux are well-known in the art and may be used or routinely Calcium Fluxin modified to assess the ability of polypeptides of the invention(including antibodies and agonists or pancreatic beta antagonists of theinvention) to mobilize calcium. For example, the FLPR assay may be usedto cells. measure influx of calcium. Cells normally have very lowconcentrations of cytosolic calcium compared to much higherextracellular calcium. Extracellular factors can cause an influx ofcalcium, leading to activation of calcium responsive signaling pathwaysand alterations in cell functions. Exemplary assays that may be used orroutinely modified to measure calcium flux by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 75 HHGCS78 279 Regulation of Assays for the regulation oftranscription through the DMEF1 response element are well-known intranscription via the art and may be used or routinely modified toassess the ability of polypeptides of the invention DMEF1 response(including antibodies and agonists or antagonists of the invention) toactivate the DMEF1 response element in element in a reporter construct(such as that containing the GLUT4 promoter) and to regulate insulinadipocytes and production. The DMEF1 response element is present in theGLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor andanother transcription factor that is required for insulin regulation ofGlut4 expression in skeletal muscle. GLUT4 is the primaryinsulin-responsive glucose transporter in fat and muscle tissue.Exemplary assays that may be used or routinely modified to test forDMEF1 response element activity (in adipocytes and pre-adipocytes) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 75HHGCS78 279 Production of Assays for measuring expression of ICAM-1 arewell-known in the art and may be used or routinely ICAM-1 modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to regulateICAM-1 expression. Exemplary assays that may be used or routinelymodified to measure ICAM-1 expression include assays disclosed in:Takacs P, et al, FASEB J, 15(2): 279-281 (2001); and, Miyamoto K, etal., Am J Pathol, 156(5): 1733-1739 (2000), the contents of each ofwhich is herein incorporated by reference in its entirety. Cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include microvascularendothelial cells (MVEC). 76 HHPSA85 280 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 77 HHSBI06 281Regulation of Caspase Apoptosis. Assays for caspase apoptosis are wellknown in the art and may be used or apoptosis in routinely modified toassess the ability of polypeptides of the invention (includingantibodies and pancreatic beta agonists or antagonists of the invention)to promote caspase protease-mediated apoptosis. cells. Apoptosis inpancreatic beta is associated with induction and progression ofdiabetes. Exemplary assays for caspase apoptosis that may be used orroutinely modified to test capase apoptosis activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in: Loweth, AC, et al., FEBSLett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int,39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4):687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001);Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBSLett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3):315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor etal., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, JAtheroscler Thromb 3(2): 75-80 (1996); the contents of each of which areherein incorporated by reference in its entirety. Pancreatic cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeRIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell linederived from a radiation induced transplantable rat islet cell tumor.The cells produce and secrete islet polypeptide hormones, and produceinsulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick etal. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad.Sci. 1980 77: 3519. 78 HHSBI65 282 Production of Assays for measuringexpression of ICAM-1 are well-known in the art and may be used orroutinely ICAM-1 modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to regulate ICAM-1 expression. Exemplary assays that may beused or routinely modified to measure ICAM-1 expression include assaysdisclosed in: Rolfe BE, et al., Atherosclerosis, 149(1): 99-110 (2000);Panettieri RA Jr, et al., J Immunol, 154(5): 2358-2365 (1995); and,Grunstein MM, et al, Am J Physiol Lung Cell Mol Physiol, 278(6):L1154-L1163 (2000), the contents of each of which is herein incorporatedby reference in its entirety. Cells that may be used according to theseassays are publicly available (e.g., through the ATCC) and/or may beroutinely generated. Exemplary cells that may be used according to theseassays include Aortic Smooth Muscle Cells (AOSMC); such as bovine AOSMC.78 HHSBI65 282 Regulation of Caspase Apoptosis. Assays for caspaseapoptosis are well known in the art and may be used or apoptosis inroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and pancreatic beta agonists orantagonists of the invention) to promote caspase protease-mediatedapoptosis. cells. Apoptosis in pancreatic beta is associated withinduction and progression of diabetes. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int. 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: # CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 79HHSGL28 283 Upregulation of HLA-DR FMAT. MHC class II is essential forcorrect presentation of antigen to CD4+ T cells. HLA-DR and Deregulationof MHC class II has been associated with autoimmune diseases (e.g.,diabetes, activation of T rheumatoid arthritis, systemic lupuserythematosis, and multiple sclerosis). Assays for cellsimmunomodulatory proteins expressed on MHC class II expressing T cellsand antigen presenting cells are well known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate the activation of T cells, and/or mediate humoralor cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of MHC class IIproducts, such as HLA-DR antigens, and the activation of T cells. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include, for example, theassays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6:138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992);Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher andZier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J HistochemCytochem 40(11): 1675-1683, the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or other- wise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 80 HISAT67 284 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 81 HJBCU75 285 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artand viability and may be used or routinely modified to assess theability of polypeptides of the invention (including proliferation ofantibodies and agonists or antagonists of the invention) to regulateviability and proliferation of pancreatic beta pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures the cells. number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely modified to test regulation of viability and proliferation ofpancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48(2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); HuglSR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), thecontents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include rat INS-1 cells. INS-1 cells are a semi-adherentcell line established from cells isolated from an X-ray induced rattransplantable insulinoma. These cells retain characteristics typical ofnative pancreatic beta cells including glucose inducible insulinsecretion. References: Asfari et al. Endocrinology 1992 130: 167. 82HJMAAO3 286 Activation of Assays for the activation of transcriptionthrough the Serum Response Element (SRE) are well- transcription knownin the art and may be used or routinely modified to assess the abilityof polypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune cells transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe (such as T-cells). polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2):105-117 (1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension culture of T cells withcytotoxic activity. 82 HJMAA03 286 Stimulation of Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 83 HKABU43 287 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 83 HKABU43 287Production of Assays for production of IL-10 and activation of T-cellsare well known in the art and may be used IL-10 and or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and activation of agonists or antagonists of theinvention) to stimulate or inhibit production of IL-10 and/or activationT-cells. of T-cells. Exemplary assays that may be used or routinelymodified to assess the ability of polypeptides and antibodies of theinvention (including agonists or antagonists of the invention) tomodulate IL-10 production and/or T-cell proliferation include, forexample, assays such as disclosed and/or cited in: Robinson, DS, et al.,“Th-2 cytokines in allergic disease” Br Med Bull; 56(4): 956-968 (2000),and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma”Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each ofwhich are herein incorporated by reference in their entirety. Exemplarycells that may be used according to these assays include Th2 cells. IL10secreted from Th2 cells may be measured as a marker of Th2 cellactivation. Th2 cells are a class of T cells that secrete IL4, IL10,IL13, IL5 and IL6. Factors that induce differentiation and activation ofTh2 cells play a major role in the initiation and pathogenesis ofallergy and asthma. Primary T helper 2 cells are generated via in vitroculture under Th2 polarizing conditions using peripheral bloodlymphocytes isolated from cord blood. 84 HKACI79 288 Activation ofAssays for the activation of transcription through the Gamma InterferonActivation Site (GAS) transcription response element are well-known inthe art and may be used or routinely modified to assess the through GASability of polypeptides of the invention (including antibodies andagonists or antagonists of the response element invention) to regulateSTAT transcription factors and modulate gene expression involved in awide in immune cells variety of cell functions. Exemplary assays fortranscription through the GAS response element that (such as T-cells).may be used or routinely modified to test GAS-response element activityof polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al.,J Immunol 155(10): 4582-4587 (1995), the contents of each of which areherein incorporated by reference in its entirety. Exemplary human Tcells, such as the SUPT cell line, that may be used according to theseassays are publicly available (e.g., through the ATCC). 84 HKACI79 288Upregulation of HLA-DR FMAT. MHC class II is essential for correctpresentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHCclass II has been associated with autoimmune diseases (e.g., diabetes,activation of rheumatoid arthritis, systemic lupus erythematosis, andmultiple sclerosis). Assays for T cells immunomodulatory proteinsexpressed on MHC class II expressing T cells and antigen presentingcells are well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to modulate theactivation of T cells, and/or mediate humoral or cell-mediated immunity.Exemplary assays that test for immunomodulatory proteins evaluate theupregulation of MHC class II products, such as HLA-DR antigens, and theactivation of T cells. Such assays that may be used or routinelymodified to test immunomodulatory activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include, for example, the assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204 (1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour etal., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, ImmunolLett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1):22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683,the contents of each of which are herein incorporated by reference inits entirety. Human T cells that may be used according to these assaysmay be isolated using techniques disclosed herein or other- wise knownin the art. Human T cells are primary human lymphocytes that mature inthe thymus and express a T Cell receptor and CD3, CD4, or CD8. Thesecells mediate humoral or cell-mediated immunity and may be preactivatedto enhance responsiveness to immunomodulatory factors. 84 HKACI79 288Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) expression isrestricted to activated T cells. CD152 is a CD152 and negative regulatorof T cell proliferation. Reduced CD152 expression has been linked toactivation of hyperproliferative and autoimmune diseases. Overexpressionof CD152 may lead to impaired T cells immunoresponses. Assays forimmunomodulatory proteins important in the maintenance of T cellhomeostasis and expressed almost exclusively on CD4+ and CD8+ T cellsare well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to modulate theactivation of T cells, maintain T cell homeostasis, and/or mediatehumoral or cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of cell surfacemarkers, such as CD152, and the activation of T cells. Such assays thatmay be used or routinely modified to test immunomodulatory activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include, for example, the assays disclosedin Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowlandet al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);McCoy et al., Immunol Cell Biol 77(1): 1-10 (1999); Oostervegal et al.,Curr Opin Immunol 11(3): 294-300 (1999); and Saito T, Curr Opin Immunol10(3): 313-321 (1998), the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or otherwise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness toimmunomodulatory factors. 85 HKIXC44 289 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transfonned with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 86 HKTAB41 290 Insulin Secretion Assays for measuring secretion ofinsulin are well-known in the art and may be used or routinely modifiedto assess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to stimulateinsulin secretion. For example, insulin secretion is measured by FMATusing anti-rat insulin antibodies. Insulin secretion from pancreaticbeta cells is upregulated by glucose and also by certainproteins/peptides, and disregulation is a key component in diabetes.Exemplary assays that may be used or routinely modified to test forstimulation of insulin secretion (from pancreatic cells) by polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in: Shimizu, H., et al., EndocrJ, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8):1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4(1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996);and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 87 HLDQU79 291 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artand viability and may be used or routinely modified to assess theability of polypeptides of the invention (including proliferation ofantibodies and agonists or antagonists of the invention) to regulateviability and proliferation of pancreatic beta pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures the cells. number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely modified to test regulation of viability and proliferation ofpancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48(2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); HuglSR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9 (1998), thecontents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include rat INS-1 cells. INS-1 cells are a semi-adherentcell line established from cells isolated from an X-ray induced rattransplantable insulinoma. These cells retain characteristics typical ofnative pancreatic beta cells including glucose inducible insulinsecretion. References: Asfari et al. Endocrinology 1992 130: 167. 87HLDQU79 291 Activation of Assays for the activation of transcriptionthrough the Serum Response Element (SRE) are well- transcription knownin the art and may be used or routinely modified to assess the abilityof polypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune transcription through the SREthat may be used or routinely modified to test SRE activity of the cells(such polypeptides of the invention (including antibodies and agonistsor antagonists of the invention) as T-cells). include assays disclosedin Berger et al., Gene 66:1-10(1998); Cullen and MaIm, Methods inEnzymol 216:362-368 (1992); Henthom et al., Proc Natl Acad Sci USA85:6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117(1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension culture of T cells withcytotoxic activity. 88 HLHAP05 292 Production of MIP-1alpha FMAT. Assaysfor immunomodulatory proteins produced by activated dendritic cellsMIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis arewell known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to mediate immunomodulation,modulate chemotaxis, and modulate T cell differentiation. Exemplaryassays that test for immunomodulatory proteins evaluate the productionof chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a),and the activation of monocytes/macrophages and T cells. Such assaysthat may be used or routinely modified to test immunomodulatory andchemotaxis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999);Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001);Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., JImmunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65:822-828 (1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 88 HLHAP05 292 Regulation of Assays for theregulation of transcription through the FAS promoter element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughthe FAS (including antibodies and agonists or antagonists of theinvention) to activate the FAS promoter promoter element element in areporter construct and to regulate transcription of FAS, a key enzymefor lipogenesis. in hepatocytes FAS promoter is regulated by manytranscription factors including SREBP. Insulin increases FAS genetranscription in livers of diabetic mice. This stimulation oftranscription is also somewhat glucose dependent. Exemplary assays thatmay be used or routinely modified to test for FAS promoter elementactivity (in hepatocytes) by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8):3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999);Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Hepatocytes that may be usedaccording to these assays, such as H4IIE cells, are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplaryhepatocytes that may be used according to these assays include rat liverhepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMPderivatives. 89 HLHCS23 293 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artand viability and may be used or routinely modified to assess theability of polypeptides of the invention (including proliferation ofantibodies and agonists or antagonists of the invention) to regulateviability and proliferation of pancreatic beta pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures the cells. number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely modified to test regulation of viability and proliferation ofpancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Ohtani KI, et al., Endocrinology, 139(1): 172-8 (1998);Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1): 29-34 (1999),the contents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include HITT15 Cells. HITT15 are an adherent epithelialcell line established from Syrian hamster islet cells transformed withSV40. These cells express glucagon, somatostatin, and glucocorticoidreceptors. The cells secrete insulin, which is stimulated by glucose andglucagon and suppressed by somatostatin or glucocorticoids. ATTC#CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre etal. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 89 HLHCS23 293Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune transcription through the SREthat may be used or routinely modified to test SRE activity of the cells(such polypeptides of the invention (including antibodies and agonistsor antagonists of the invention) as T-cells). include assays disclosedin Berger et al., Gene 66:1-10 (1998); Cullen and MaIm, Methods inEnzymol 2 16:362-368 (1992); Henthom et al., Proc NatI Acad Sci USA85:6342-6346 (1988); and Black et al., Virus Genes 12(2): 105-117(1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CuLL cellline, which is an IL-2 dependent suspension culture ofT cells withcytotoxic activity 89 HLHCS23 293 Production of IFNgamma FMAT. IFNgplays a central role in the immune system and is considered to be aIFNgamma using proinflammatory cytokine. IFNg promotes TH1 and inhibitsTH2 differentiation; promotes IgG2a a T cells and inhibits IgEsecretion; induces macrophage activation; and increases MHC expression.Assays for immunomodulatory proteins produced by T cells and NK cellsthat regulate a variety of inflammatory activities and inhibit TH2helper cell functions are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation, regulate inflammatoryactivities, modulate TH2 helper cell function, and/or mediate humoral orcell- mediated immunity. Exemplary assays that test for immunomodulatoryproteins evaluate the production of cytokines, such as Interferon gamma(IFNg), and the activation of T cells. Such assays that may be used orroutinely modified to test immunomodulatory activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Miraglia et al., JBiomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes:a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J ClinLab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856:22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), andRheumatology (Oxford) 38(3): 214-20 (1999), the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or otherwise known in the art. Human T cellsare primary human lymphocytes that mature in the thymus and express a TCell receptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 90 HLICE88 294 Activation of Assays for theactivation of transcription through the Serum Response Element (SRE) arewell- transcription known in the art and may be used or routinelymodified to assess the ability of polypeptides of the through seruminvention (including antibodies and agonists or antagonists of theinvention) to regulate the serum response element response factors andmodulate the expression of genes involved in growth. Exemplary assaysfor in immune cells transcription through the SRE that may be used orroutinely modified to test SRE activity of the (such as T-cells).polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension culture of T cells with cytotoxic activity. 90 HLICE88 294Stimulation of Assays for measuring secretion of insulin are well-knownin the art and may be used or routinely insulin secretion modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or from pancreatic antagonists of the invention)to stimulate insulin secretion. For example, insulin secretion is betacells. measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ahren, B., etal., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al.,Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett,377(2): 237-9 (1995); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 91 HLMGP50 295 Upregulation of HLA-DR FMAT.MHC class II is essential for correct presentation of antigen to CD4+ Tcells. HLA-DR and Deregulation of MHC class II has been associated withautoimmune diseases (e.g., diabetes, activation of rheumatoid arthritis,systemic lupus erythematosis, and multiple sclerosis). Assays for Tcells immunomodulatory proteins expressed on MHC class II expressing Tcells and antigen presenting cells are well known in the art and may beused or routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate the activation of T cells, and/or mediate humoralor cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of MHC class IIproducts, such as HLA-DR antigens, and the activation of T cells. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include, for example, theassays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6:138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992);Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher andZier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J HistochemCytochem 40(11): 1675-1683, the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or other- wise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 92 HLMMX62 296 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 93 HLQCX36 297Proliferation of Assays for the regulation (i.e. increases or decreases)of viability and proliferation of cells in vitro immune cells arewell-known in the art and may be used or routinely modified to assessthe ability of polypeptides (such as the of the invention (includingantibodies and agonists or antagonists of the invention) to regulateHMC-1 human viability and proliferation of eosinophil cells and celllines. For example, the CellTiter-Gloô mast cell line) Luminescent CellViability Assay (Promega Corp., Madison, WI, USA ) can be used tomeasure the number of viable cells in culture based on quantitation ofthe ATP present which signals the presence of metabolically activecells. Mast cells are found in connective and mucosal tissues throughoutthe body. Mast cell activation (via immunoglobulin E -antigen, promotedby T helper cell type 2 cytokines) is an important component of allergicdisease. Dysregulation of mast cell apoptosis may play a role inallergic disease and mast cell tumor survival. Mast cell lines that maybe used according to these assays are publicly available and/or may beroutinely generated. Exemplary mast cells that may be used according tothese assays include HMC-1, which is an immature human mast cell lineestablished from the peripheral blood of a patient with mast cellleukemia, and exhibits many characteristics of immature mast cells. 93HLQCX36 297 Activation of Kinase assay. Kinase assays, for example anGSK-3 kinase assay, for PI3 kinase signal Skeletal Mucle transductionthat regulate glucose metabolism and cell survivial are well-known inthe art and may Cell PI3 Kinase be used or routinely modified to assessthe ability of polypeptides of the invention (including Signallingantibodies and agonists or antagonists of the invention) to promote orinhibit glucose metabolism Pathway and cell survival. Exemplary assaysfor PI3 kinase activity that may be used or routinely modified to testPI3 kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al.,Diabetes 48(8): 1662-1666 (1999), the contents of each of which areherein incorporated by reference in its entirety. Rat myoblast cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 94 HLWAF06 298 Activation of Kinaseassay. Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110(1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 95 HLWDB73 299Activation of Kinase assay. Kinase assays, for examplek Elk-1 kinaseassays, for ERK signal transduction that Skeletal Muscle regulate cellproliferation or differentiation are well known in the art and may beused or routinely Cell ERK modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orSignalling antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Pathway Exemplary assaysfor ERK kinase activity that may be used or routinely modified to testERK kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Rat myoblast cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC). Exemplary rat myoblast cells that may be used according tothese assays include L6 cells. L6 is an adherent rat myoblast cell line,isolated from primary cultures of rat thigh muscle, that fuses to formmultinucleated myotubes and striated fibers after culture indifferentiation media. 96 HLYDF73 300 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 97 HLYGB19 301 Activation of Kinase assay. Kinase assays, forexample an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucletransduction that regulate glucose metabolism and cell survivial arewell-known in the art and may Cell PI3 Kinase be used or routinelymodified to assess the ability of polypeptides of the invention(including Signalling antibodies and agonists or antagonists of theinvention) to promote or inhibit glucose metabolism Pathway and cellsurvival. Exemplary assays for PI3 kinase activity that may be used orroutinely modified to test PI3 kinase-induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by reference in itsentirety. Rat myoblast cells that may be used according to these assaysare publicly available (e.g., through the ATCC). Exemplary rat myoblastcells that may be used according to these assays include L6 cells. L6 isan adherent rat myoblast cell line, isolated from primary cultures ofrat thigh muscle, that fuses to form multinucleated myotubes andstriated fibers after culture in differentiation media. 97 HLYGB19 301Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) expression isrestricted to activated T cells. CD152 is a CD152 and negative regulatorof T cell proliferation. Reduced CD152 expression has been linked toactivation of hyperproliferative and autoimmune diseases. Overexpressionof CD152 may lead to impaired T cells immunoresponses. Assays forimmunomodulatory proteins important in the maintenance of T cellhomeostasis and expressed almost exclusively on CD4+ and CD8+ T cellsare well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to modulate theactivation of T cells, maintain T cell homeostasis, and/or mediatehumoral or cell-mediated immunity. Exemplary assays that test forimmunomodulatory proteins evaluate the upregulation of cell surfacemarkers, such as CD152, and the activation of T cells. Such assays thatmay be used or routinely modified to test immunomodulatory activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include, for example, the assays disclosedin Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowlandet al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);McCoy et al., Immunol Cell Biol 77(1): 1-10 (1999); Oostervegal et al.,Curr Opin Immunol 11(3): 294-300 (1999); and Saito T, Curr Opin Immunol10(3): 313-321 (1998), the contents of each of which are hereinincorporated by reference in its entirety. Human T cells that may beused according to these assays may be isolated using techniquesdisclosed herein or otherwise known in the art. Human T cells areprimary human lymphocytes that mature in the thymus and express a T Cellreceptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness toimmunomodulatory factors. 98 HLYGY91 302 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 98 HLYGY91 302 Production of Assays for production of IL-13 andactivation of T-cells are well known in the art and may be used IL-13and or routinely modified to assess the ability of polypeptides of theinvention (including antibodies and activation of agonists orantagonists of the invention) to stimulate or inhibit production ofIL-13 and/or activation T-cells. of T-cells. Exemplary assays for IL-13production that may be used or routinely modified to test activity ofpolypeptides and antibodies of the invention (including agonists orantagonists of the invention) include, for example, assays such asdisclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13independently of IL-4 in Experimental asthma” Science; 282: 2261-2263(1998), and Wills-Karp M, et al., “Interleukin-13: central mediator ofallergic asthma” Science; 282: 2258-2261 (1998); the contents of each ofwhich are herein incorporated by reference in their entirety. Exemplarycells that may be used according to these assays include Th2 cells.IL13, a Th2 type cytokine, is a potent stimulus for mucus production,airway hyper-responsiveness and allergic asthma. Th2 cells are a classof T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors thatinduce differentiation and activation of Th2 cells play a major role inthe initiation and pathogenesis of allergy and asthma. Primary T helper2 cells are generated in in vitro culture under Th2 polarizingconditions using peripheral blood lymphocytes isolated from cord blood.99 HMDAB29 303 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ohtani KI, etal., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp ClinEndocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 99 HMDAB29 303 Insulin SecretionAssays for measuring secretion of insulin are well-known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 99 HMDAB29 303 Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4)expression is restricted to activated T cells. CD152 is a CD152 andnegative regulator of T cell proliferation. Reduced CD152 expression hasbeen linked to activation of hyperproliferative and autoimniunediseases. Overexpression of CD152 may lead to impaired T cellsimmunoresponses. Assays for immunomodulatory proteins important in themaintenance of T cell homeostasis and expressed almost exclusively onCD4+ and CD8+ T cells are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate the activation of T cells, maintain T cellhomeostasis, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof cell surface markers, such as CD152, and the activation of T cells.Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); McCoy et al., Immunol Cell Biol 77(1): 1-10 (1999); Oostervegal et al., Curr Opin Immunol 11(3): 294-300(1999); and Saito T, Curr Opin Immunol 10(3): 313-321 (1998), thecontents of each of which are herein incorporated by reference in itsentirety. Human T cells that may be used according to these assays maybe isolated using techniques disclosed herein or otherwise known in theart. Human T cells are primary human lymphocytes that mature in thethymus and express a T Cell receptor and CD3, CD4, or CD8. These cellsmediate humoral or cell- mediated immunity and may be preactivated toenhance responsiveness to immunomodulatory factors. 100 HMDAD44 304Regulation of Assays for the regulation of transcription through theDMEF1 response element are well-known in transcription via the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention DMEF1 response (including antibodies and agonists orantagonists of the invention) to activate the DMEF1 response element inelement in a reporter construct (such as that containing the GLUT4promoter) and to regulate insulin adipocytes and production. The DMEF1response element is present in the GLUT4 promoter and binds to MEF2pre-adipocytes transcription factor and another transcription factorthat is required for insulin regulation of Glut4 expression in skeletalmuscle. GLUT4 is the primary insulin-responsive glucose transporter infat and muscle tissue. Exemplary assays that may be used or routinelymodified to test for DMEF1 response element activity (in adipocytes andpre-adipocytes) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedinThai, M. V., et al., J Biol Chem, 273(23): 14285-92 (1998); Mora, S.,et al., J Biol Chem, 275(21): 16323-8 (2000); Liu, M. L., et al., J BiolChem, 269(45): 28514-21 (1994); “Identification of a 30-base pairregulatory element and novel DNA binding protein that regulates thehuman GLUT4 promoter in transgenic mice”, J Biol Chem. 2000 Aug 4;275(31): 23666-73; Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Adipocytes and pre-adipocytes that may be used according to these assaysare publicly available (e.g., through the ATCC) and/or may be routinelygenerated. Exemplary cells that may be used according to these assaysinclude the mouse 3T3-L1 cell line which is an adherent mousepreadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of3T3 fibroblasts developed through clonal isolation. These cells undergoa pre-adipocyte to adipose-like conversion under appropriatedifferentiation culture conditions. 100 HMDAD44 304 Production of TNFaFMAT. Assays for immunomodulatory proteins produced by activatedmacrophages, T cells, TNF alpha by fibroblasts, smooth muscle, and othercell types that exert a wide variety of inflammatory and dendritic cellscytotoxic effects on a variety of cells are well known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to mediate immunomodulation, modulate inflammation andcytotoxicity. Exemplary assays that test for immunomodulatory proteinsevaluate the production of cytokines such as tumor necrosis factor alpha(TNFa), and the induction or inhibition of an inflammatory or cytotoxicresponse. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999);Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198);Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., JImmunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65:822-828 (1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 100 HMDAD44 304 Production of MCP-1 FMAT. Assaysfor immunomodulatory proteins that are produced by a large variety ofcells MCP-1 and act to induce chemotaxis and activation of monocytes andT cells are well known in the art and may be used or routinely modifiedto assess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to mediateimmunomodulation, induce chemotaxis, and modulate immune cellactivation. Exemplary assays that test for immunomodulatory proteinsevaluate the production of cell surface markers, such as monocytechemoattractant protein (MCP), and the activation of monocytes and Tcells. Such assays that may be used or routinely modified to testimmunomodulatory and diffferentiation activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); Satthaporn and Eremin, J R CollSurg Ednb 45(1): 9-19 (2001); and Verhasselt et al., J Immunol 158:2919-2925 (1997), the contents of each of which are herein incorporatedby reference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 101 HMEDI90 305 Regulation of Assays for theregulation of transcription of Malic Enzyme are well-known in the artand may be transcription of used or routinely modified to assess theability of polypeptides of the invention (including Malic Enzyme inantibodies and agonists or antagonists of the invention) to regulatetranscription of Malic Enzyme, a adipocytes key enzyme in lipogenesis.Malic enzyme is involved in lipogenesisand its expression is stimultedby insulin. ME promoter contains two direct repeat (DR1)- like elementsMEp and MEd identified as putative PPAR response elements. ME promotermay also responds to AP1 and other transcription factors. Exemplaryassays that may be used or routinely modified to test for regulation oftranscription of Malic Enzyme (in adipoocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Streeper, R. S., et al., MolEndocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., MolEndocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem,274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32):20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Hepatocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assaysincludes the H4IIE rat liver hepatoma cell line. 102 HMIBF07 306Production of MCP-1 FMAT. Assays for immunomodulatory proteins that areproduced by a large variety of cells MCP-1 and act to induce chemotaxisand activation of monocytes and T cells are well known in the art andmay be used or routinely modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) to mediate immunomodulation, induce chemotaxis, andmodulate immune cell activation. Exemplary assays that test forimmunomodulatory proteins evaluate the production of cell surfacemarkers, such as monocyte chemoattractant protein (MCP), and theactivation of monocytes and T cells. Such assays that may be used orroutinely modified to test immunomodulatory and diffferentiationactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inMiraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland etal., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001); andVerhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of eachof which are herein incorporated by reference in its entirety. Humandendritic cells that may be used according to these assays may beisolated using techniques disclosed herein or otherwise known in theart. Human dendritic cells are antigen presenting cells in suspensionculture, which, when activated by antigen and/or cytokines, initiate andupregulate T cell proliferation and functional activities. 102 HMIBF07306 Insulin Secretion Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to stimulate insulinsecretion. For example, insulin secretion is measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9(2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999);Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L.K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et.al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contentsof each of which is herein incorporated by reference in its entirety.Pancreatic cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary pancreatic cells that may be used according to these assaysinclude HITT15 Cells. HITT15 are an adherent epithelial cell lineestablished from Syrian hamster islet cells transformed with SV40. Thesecells express glucagon, somatostatin, and glucocorticoid receptors. Thecells secrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 102 HMIBF07 306 Stimulation ofAssays for measuring secretion of insulin are well-known in the art andmay be used or routinely insulin secretion modified to assess theability of polypeptides of the invention (including antibodies andagonists or from pancreatic antagonists of the invention) to stimulateinsulin secretion. For example, insulin secretion is beta cells.measured by FMAT using anti-rat insulin antibodies. Insulin secretionfrom pancreatic beta cells is upregulated by glucose and also by certainproteins/peptides, and disregulation is a key component in diabetes.Exemplary assays that may be used or routinely modified to test forstimulation of insulin secretion (from pancreatic cells) by polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in: Ahren, B., et al., Am JPhysiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology,138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9(1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4:193-204 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 103 HMICP65 307 Production of Assays formeasuring expression of VCAM are well-known in the art and may be usedor routinely VCAM in modified to assess the ability of polypeptides ofthe invention (including antibodies and agonists or endothelial cellsantagonists of the invention) to regulate VCAM expression. For example,FMAT may be used to (such as human meaure the upregulation of cellsurface VCAM-1 expresssion in endothelial cells. Endothelial cellsumbilical vein are cells that line blood vessels, and are involved infunctions that include, but are not limited to, endothelial cellsangiogenesis, vascular permeability, vascular tone, and immune cellextravasation. Exemplary (HUVEC)) endothelial cells that may be usedaccording to these assays include human umbilical vein endothelial cells(HUVEC), which are available from commercial sources. The expression ofVCAM (CD106), a membrane-associated protein, can be upregulated bycytokines or other factors, and contributes to the extravasation oflymphocytes, leucocytes and other immune cells from blood vessels; thusVCAM expression plays a role in promoting immune and inflammatoryresponses. 103 HMICP65 307 Activation of Assays for the activation oftranscription through the Gamma Interferon Activation Site (GAS)transcription response element are well-known in the art and may be usedor routinely modified to assess the through GAS ability of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe response element invention) to regulate STAT transcription factorsand modulate gene expression involved in a wide in immune cells varietyof cell functions. Exemplary assays for transcription through the GASresponse element that (such as T-cells). may be used or routinelymodified to test GAS-response element activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainenet al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol155(10): 4582-4587 (1995), the contents of each of which are hereinincorporated by reference in its entirety. Exemplary human T cells, suchas the SUPT cell line, that may be used according to these assays arepublicly available (e.g., through the ATCC). 103 HMICP65 307Upregulation of HLA-DR FMAT. MHC class II is essential for correctpresentation of antigen to CD4+ T cells. HLA-DR and Deregulation of MHCclass II has been associated with autoimmune diseases (e.g., diabetes,activation of rheumatoid arthritis, systemic lupus erythematosis, andmultiple sclerosis). Assays for T cells immunomodulatory proteinsexpressed on MHC class II expressing T cells and antigen presentingcells are well known in the art and may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to modulate theactivation of T cells, and/or mediate humoral or cell-mediated immunity.Exemplary assays that test for immunomodulatory proteins evaluate theupregulation of MHC class II products, such as HLA-DR antigens, and theactivation of T cells. Such assays that may be used or routinelymodified to test immunomodulatory activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include, for example, the assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204 (1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour etal., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, ImmunolLett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1):22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683,the contents of each of which are herein incorporated by reference inits entirety. Human T cells that may be used according to these assaysmay be isolated using techniques disclosed herein or other- wise knownin the art. Human T cells are primary human lymphocytes that mature inthe thymus and express a T Cell receptor and CD3, CD4, or CD8. Thesecells mediate humoral or cell-mediated immunity and may be preactivatedto enhance responsiveness to immunomodulatory factors. 104 HMSHC86 308Activation of Kinase assay. Kinase assays, for examplek Elk-i kinaseassays, for ERK signal transduction that Skeletal Muscle regulate cellproliferation or differentiation are well known in the art and may beused or routinely Cell ERK modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orSignalling antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Pathway Exemplary assaysfor ERK kinase activity that may be used or routinely modified to testERK kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Rat myoblast cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC). Exemplary rat myoblast cells that may be used according tothese assays include L6 cells. L6 is an adherent rat myoblast cell line,isolated from primary cultures of rat thigh muscle, that fuses to formmultinucleated myotubes and striated fibers after culture indifferentiation media. 105 HMUAN45 309 Stimulation of Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 106 HMVBC31 310 Stimulation of Assays formeasuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 107 HMWBL03 311 Regulation of Caspase Apoptosis. Assays forcaspase apoptosis are well known in the art and may be used or apoptosisin routinely modified to assess the ability of polypeptides of theinvention (including antibodies and pancreatic beta agonists orantagonists of the invention) to promote caspase protease-mediatedapoptosis. cells. Apoptosis in pancreatic beta is associated withinduction and progression of diabetes. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 108HNFGR08 312 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: FriedrichsenBN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al.,Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998Jul 10; 273(28): 17771-9 (1998), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 109 HNGAK51 313 Insulin Secretion Assaysfor measuring secretion of insulin are well-known in the art and may beused or routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 110 HNGDX18 314 Regulation of Assays for the regulation oftranscription through the DMEF1 response element are well-known intranscription via the art and may be used or routinely modified toassess the ability of polypeptides of the invention DMEF1 response(including antibodies and agonists or antagonists of the invention) toactivate the DMEF1 response element in element in a reporter construct(such as that containing the GLUT4 promoter) and to regulate insulinadipocytes and production. The DMEF1 response element is present in theGLUT4 promoter and binds to MEF2 pre-adipocytes transcription factor andanother transcription factor that is required for insulin regulation ofGlut4 expression in skeletal muscle. GLUT4 is the primaryinsulin-responsive glucose transporter in fat and muscle tissue.Exemplary assays that may be used or routinely modified to test forDMEF1 response element activity (in adipocytes and pre-adipocytes) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 110HNGDX18 314 Activation of Assays for the activation of transcriptionthrough the Gamma Interferon Activation Site (GAS) transcriptionresponse element are well-known in the art and may be used or routinelymodified to assess the through GAS ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theresponse element invention) to regulate STAT transcription factors andmodulate gene expression involved in a wide in immune cells variety ofcell functions. Exemplary assays for transcription through the GASresponse element that (such as T-cells). may be used or routinelymodified to test GAS-response element activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainenet al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol155(10): 4582-4587 (1995), the contents of each of which are hereinincorporated by reference in its entirety. Exemplary mouse T cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary T cells that may be used according to theseassays include the CTLL cell line, which is a suspension culture of IL-2dependent cytotoxic T cells. 110 HNGDX18 314 Activation of Assays forthe activation of transcription through the Serum Response Element (SRE)are well- transcription known in the art and may be used or routinelymodified to assess the ability of polypeptides of the through seruminvention (including antibodies and agonists or antagonists of theinvention) to regulate the serum response element response factors andmodulate the expression of genes involved in growth. Exemplary assaysfor in immune cells transcription through the SRE that may be used orroutinely modified to test SRE activity of the (such as T-cells).polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension culture of T cells with cytotoxic activity. 111 HNGGP65 315Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Hepatocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4):479-500 (1999); the contents of each of which are herein incorporated byreference in its entirety. Rat liver hepatoma cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary rat liver hepatoma cells that may be used according tothese assays include H4lle cells, which are known to respond toglucocorticoids, insulin, or cAMP derivatives. 112 HNGIV64 316Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 112 HNGIV64 316 Productionof Assays for production of IL-10 and activation of T-cells are wellknown in the art and may be used IL-10 and or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and activation of agonists or antagonists of the invention)to stimulate or inhibit production of IL-10 and/or activation T-cells.of T-cells. Exemplary assays that may be used or routinely modified toassess the ability of polypeptides and antibodies of the invention(including agonists or antagonists of the invention) to modulate IL-10production and/or T-cell proliferation include, for example, assays suchas disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines inallergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al.,“T-helper type 2 cell-directed therapy for asthma” Pharmacology &Therapeutics; 88: 187-196 (2000); the contents of each of which areherein incorporated by reference in their entirety. Exemplary cells thatmay be used according to these assays include Th2 cells. IL10 secretedfrom Th2 cells may be measured as a marker of Th2 cell activation. Th2cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6.Factors that induce differentiation and activation of Th2 cells play amajor role in the initiation and pathogenesis of allergy and asthma.Primary T helper 2 cells are generated via in vitro culture under Th2polarizing conditions using peripheral blood lymphocytes isolated fromcord blood. 113 HNGMW45 317 Regulation of Assays for the regulation oftranscription through the PEPCK promoter are well-known in the arttranscription and may be used or routinely modified to assess theability of polypeptides of the invention through the (includingantibodies and agonists or antagonists of the invention) to activate thePEPCK promoter PEPCK promoter in a reporter construct and regulate livergluconeogenesis. Exemplary assays for regulation of in hepatocytestranscription through the PEPCK promoter that may be used or routinelymodified to test for PEPCK promoter activity (in hepatocytes) ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., JBiol Chem 275(23): 17814-17820 (2000), the contents of each of which isherein incorporated by reference in its entirety. Hepatocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary liverhepatoma cells that may be used according to these assays include H4llecells, which contain a tyrosine amino transferase that is inducible withglucocorticoids, insulin, or cAMP derivatives. 114 HNGPJ25 318Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to regulate STAT transcription factors and modulategene expression involved in a wide in immune cells variety of cellfunctions. Exemplary assays for transcription through the GAS responseelement that (such as T-cells). may be used or routinely modified totest GAS-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6):1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587(1995), the contents of each of which are herein incorporated byreference in its entirety. Exemplary mouse T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary T cells that may be used according to these assaysinclude the CTLL cell line, which is a suspension culture of IL-2dependent cytotoxic T cells. 114 HNGPJ25 318 Activation of Assays forthe activation of transcription through the Serum Response Element (SRE)are well- transcription known in the art and may be used or routinelymodified to assess the ability of polypeptides of the through seruminvention (including antibodies and agonists or antagonists of theinvention) to regulate the serum response element response factors andmodulate the expression of genes involved in growth. Exemplary assaysfor in immune cells transcription through the SRE that may be used orroutinely modified to test SRE activity of the (such as T-cells).polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension culture of T cells with cytotoxic activity. 114 HNGPJ25 318Regulation of Assays for the regulation of transcription of Malic Enzymeare well-known in the art and may be transcription of used or routinelymodified to assess the ability of polypeptides of the invention(including Malic Enzyme in antibodies and agonists or antagonists of theinvention) to regulate transcription of Malic Enzyme, a adipocytes keyenzyme in lipogenesis. Malic enzyme is involved in lipogenesisand itsexpression is stimulted by insulin. ME promoter contains two directrepeat (DR1)- like elements MEp and MEd identified as putative PPARresponse elements. ME promoter may also responds to AP1 and othertranscription factors. Exemplary assays that may be used or routinelymodified to test for regulation of transcription of Malic Enzyme (inadipoocytes) by polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed in:Streeper, R. S., et al, Mol Endocrinol, 12(11): 1778-91 (1998);Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994);Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg,A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al.,Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216:362-368 (1992), the contents of each of which is herein incorporated byreference in its entirety. Hepatocytes that may be used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary hepatocytes that may be used accordingto these assays includes the H4IIE rat liver hepatoma cell line. 115HNHEN82 319 Activation of Kinase assay. Kinase assays, for example anGSK-3 assays, for PI3 kinase signal transduction that Adipocyte PI3regulate glucose metabolism and cell survival are well-known in the artand may be used or Kinase Signalling routinely modified to assess theability of polypeptides of the invention (including antibodies andPathway agonists or antagonists of the invention) to promote or inhibitglucose metabolism and cell survival. Exemplary assays for PI3 kinaseactivity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina etal., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes48(8): 1662-1666 (1999), the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3- L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 116 HNHFE71 320 Activationof Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3kinase signal transduction that Adipocyte PI3 regulate glucosemetabolism and cell survival are well-known in the art and may be usedor Kinase Signalling routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and Pathway agonistsor antagonists of the invention) to promote or inhibit glucosemetabolism and cell survival. Exemplary assays for PI3 kinase activitythat may be used or routinely modified to test PI3 kinase- inducedactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inForrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al.,Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8):1662-1666 (1999), the contents of each of which are herein incorporatedby reference in its entirety. Mouse adipocyte cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse adipocyte cells that may be used according tothese assays include 3T3- L1 cells. 3T3-L1 is an adherent mousepreadipocyte cell line that is a continous substrain of 3T3 fibroblastcells developed through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 116 HNHFE71 320 Production of Assays for measuringexpression of VCAM are well-known in the art and may be used orroutinely VCAM in modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or endothelial cellsantagonists of the invention) to regulate VCAM expression. For example,FMAT may be used to (such as human meaure the upregulation of cellsurface VCAM-1 expresssion in endothelial cells. Endothelial cellsumbilical vein are cells that line blood vessels, and are involved infunctions that include, but are not limited to, endothelial cellsangiogenesis, vascular permeability, vascular tone, and immune cellextravasation. Exemplary (HUVEC)) endothelial cells that may be usedaccording to these assays include human umbilical vein endothelial cells(HUVEC), which are available from commercial sources. The expression ofVCAM (CD106), a membrane-associated protein, can be upregulated bycytokines or other factors, and contributes to the extravasation oflymphocytes, leucocytes and other immune cells from blood vessels; thusVCAM expression plays a role in promoting immune and inflammatoryresponses. 116 HNHFE71 320 Calcium flux in Assays for measuring calciumflux are well-known in the art and may be used or routinely modifiedimmune cells to assess the ability of polypeptides of the invention(including antibodies and agonists or (such as antagonists of theinvention) to mobilize calcium. Cells normally have very lowconcentrations of monocytes) cytosolic calcium compared to much higherextracellular calcium. Extracellular factors can cause an influx ofcalcium, leading to activation of calcium responsive signaling pathwaysand alterations in cell functions. Exemplary assays that may be used orroutinely modified to measure calcium flux in immune cells (such asmonocytes) include assays disclosed in: Chan, CC, et al., J PharmacolExp Ther, 269(3): 891-896 (1994); Andersson, K, et al., Cytokine,12(12): 1784-1787 (2000); Scully, SP, et al., J Clin Invest, 74(2)589-599 (1984); and, Sullivan, E, et al., Methods Mol Biol, 114: 125-133(1999), the contents of each of which is herein incorporated byreference in its entirety. Cells that may be used according to theseassays are publicly available (e.g., through the ATCC) and/or may beroutinely generated. Exemplary cells that may be used according to theseassays include the THP-1 monocyte cell line. 117 HNHKV56 321 Regulationof Assays for the regulation of viability and proliferation of cells invitro are well-known in the art and viability and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1):172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1):29-34 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 118 HOACG07 322 Stimulation of Assays for measuring calcium fluxare well-known in the art and may be used or routinely Calcium Flux inmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or pancreatic beta antagonists of theinvention) to mobilize calcium. For example, the FLPR assay may be usedto cells. measure influx of calcium. Cells normally have very lowconcentrations of cytosolic calcium compared to much higherextracellular calcium. Extracellular factors can cause an influx ofcalcium, leading to activation of calcium responsive signaling pathwaysand alterations in cell functions. Exemplary assays that may be used orroutinely modified to measure calcium flux by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 119 HODBB70 323 Activation of Assays for the activation oftranscription through the cAMP response element are well-known intranscription the art and may be used or routinely modified to assessthe ability of polypeptides of the invention through cAMP (includingantibodies and agonists or antagonists of the invention) to increasecAMP and regulate response element CREB transcription factors, andmodulate expression of genes involved in a wide variety of cell inimmune cells functions. Exemplary assays for transcription through thecAMP response element that may be used (such as T-cells). or routinelymodified to test cAMP-response element activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Black etal., Virus Genes 15(2): 105-117 (1997); and Belkowski et al., J Immunol161(2): 659-665 (1998), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse T cells that may be used according to theseassays include the CTLL cell line, which is a suspension culture of IL-2dependent cytotoxic T cells. 119 HODBB70 323 Regulation of Assays forthe regulation of transcription through the PEPCK promoter arewell-known in the art transcription and may be used or routinelymodified to assess the ability of polypeptides of the invention throughthe (including antibodies and agonists or antagonists of the invention)to activate the PEPCK promoter PEPCK promoter in a reporter constructand regulate liver gluconeogenesis. Exemplary assays for regulation ofin hepatocytes transcription through the PEPCK promoter that may be usedor routinely modified to test for PEPCK promoter activity (inhepatocytes) of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Lochhead et al., Diabetes 49(6): 896-903 (2000); andYeagley et al., J Biol Chem 275(23): 17814-17820 (2000), the contents ofeach of which is herein incorporated by reference in its entirety.Hepatocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary liver hepatoma cells that may be used according to theseassays include H4lle cells, which contain a tyrosine amino transferasethat is inducible with glucocorticoids, insulin, or cAMP derivatives.120 HOEBK60 324 Insulin Secretion Assays for measuring secretion ofinsulin are well-known in the art and may be used or routinely modifiedto assess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to stimulateinsulin secretion. For example, insulin secretion is measured by FMATusing anti-rat insulin antibodies. Insulin secretion from pancreaticbeta cells is upregulated by glucose and also by certainproteins/peptides, and disregulation is a key component in diabetes.Exemplary assays that may be used or routinely modified to test forstimulation of insulin secretion (from pancreatic cells) by polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in: Shimizu, H., et al., EndocrJ, 47(3): 261-9 (2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8):1305-17 (1999); Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4(1998); Olson, L. K., et al., J Biol Chem, 271(28): 16544-52 (1996);and, Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 121 HOFAA78 325 Activation of Kinase assay. Kinase assays, forexample an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucletransduction that regulate glucose metabolism and cell survivial arewell-known in the art and may Cell PI3 Kinase be used or routinelymodified to assess the ability of polypeptides of the invention(including Signalling antibodies and agonists or antagonists of theinvention) to promote or inhibit glucose metabolism Pathway and cellsurvival. Exemplary assays for PI3 kinase activity that may be used orroutinely modified to test PI3 kinase-induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by ref- erence in itsentirety. Rat myoblast cells that may be used according to these assaysare publicly available (e.g., through the ATCC). Exemplary rat myoblastcells that may be used according to these assays include L6 cells. L6 isan adherent rat myoblast cell line, isolated from primary cultures ofrat thigh muscle, that fuses to form multinucleated myotubes andstriated fibers after culture in differentiation media. 122 HOFNB74 326Insulin Secretion Assays for measuring secretion of insulin arewell-known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to stimulate insulinsecretion. For example, insulin secretion is measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Shimizu, H., et al., Endocr J, 47(3): 261-9(2000); Salapatek, A. M., et al., Mol Endocrinol, 13(8): 1305-17 (1999);Filipsson, K., et al., Ann N Y Acad Sci, 865: 441-4 (1998); Olson, L.K., et al., J Biol Chem, 271(28): 16544-52 (1996); and, Miraglia S et.al., Journal of Biomolecular Screening, 4: 193-204 (1999), the contentsof each of which is herein incorporated by reference in its entirety.Pancreatic cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary pancreatic cells that may be used according to these assaysinclude HITT15 Cells. HITT15 are an adherent epithelial cell lineestablished from Syrian hamster islet cells transformed with SV40. Thesecells express glucagon, somatostatin, and glucocorticoid receptors. Thecells secrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 123 HORBS82 327 Activation of Kinaseassay. Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 123 HORBS82 327Activation of Kinase assay. JNK kinase assays for signal transductionthat regulate cell proliferation, activation, JNK Signaling or apoptosisare well known in the art and may be used or routinely modified toassess the ability of Pathway in polypeptides of the invention(including antibodies and agonists or antagonists of the invention) toimmune cells promote or inhibit cell proliferation, activation, andapoptosis. Exemplary assays for JNK kinase (such as activity that may beused or routinely modified to test JNK kinase-induced activity ofpolypeptides eosinophils). of the invention (including antibodies andagonists or antagonists of the invention) include the assays disclosedin Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al.,Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64:29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and CobbMH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of eachof which are herein incorporated by reference in its entirety. Exemplarycells that may be used according to these assays include eosinophils.Eosinophils are important in the late stage of allergic reactions; theyare recruited to tissues and mediate the inflammatory response of latestage allergic reaction. Moreover, exemplary assays that may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to modulate signal transduction, cell proliferation,activation, or apoptosis in eosinophils include assays disclosed and/orcited in: Zhang JP, et al., “Role of caspases in dexamethasone-inducedapoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils” Clin Exp Immunol; Oct;122(1): 20-7 (2000); Hebestreit H, et al., “Disruption of fas receptorsignaling by nitric oxide in eosinophils” J Exp Med; Feb 2; 187(3):415-25 (1998); J Allergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74;and, Sousa AR, et al., “In vivo resistance to corticosteroids inbronchial asthma is associated with enhanced phosyphorylation of JUNN-terminal kinase and failure of prednisolone to inhibit JUN N-terminalkinase phosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74(1999); the contents of each of which are herein incorporated byreference in its entirety. 123 HORBS82 327 Regulation of CaspaseApoptosis. Assays for caspase apoptosis are well known in the art andmay be used or apoptosis of routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and immune cellsagonists or antagonists of the invention) to regulate caspaseprotease-mediated apoptosis in immune (such as mast cells (such as, forexample, in mast cells). Mast cells are found in connective and mucosaltissues cells). throughout the body, and their activation viaimmunoglobulin E -antigen, promoted by T helper cell type 2 cytokines,is an important component of allergic disease. Dysregulation of mastcell apoptosis may play a role in allergic disease and mast cell tumorsurvival. Exemplary assays for caspase apoptosis that may be used orroutinely modified to test capase apoptosis activity induced bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in: Masuda A,et al., J Biol Chem, 276(28): 26107-26113 (2001); Yeatman CF 2nd, etal., J Exp Med, 192(8): 1093-1103 (2000); Lee et al., FEBS Lett485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000);and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); thecontents of each of which are herein incorporated by reference in itsentirety. Immune cells that may be used according to these assays arepublicly available (e.g., through commercial sources). Exemplary immunecells that may be used according to these assays include mast cells suchas the HMC human mast cell line. 123 HORBS82 327 Production of Assaysfor production of IL-13 and activation of T-cells are well known in theart and may be used IL-13 and or routinely modified to assess theability of polypeptides of the invention (including antibodies andactivation of agonists or antagonists of the invention) to stimulate orinhibit production of IL-13 and/or activation T-cells. of T-cells.Exemplary assays for IL-13 production that may be used or routinelymodified to test activity of polypeptides and antibodies of theinvention (including agonists or antagonists of the invention) include,for example, assays such as disclosed and/or cited in: Grunig, G, etal., “Requirement for IL-13 independently of IL-4 in Experimentalasthma” Science; 282: 2261-2263 (1998), and Wills-Karp M, et al.,“Interleukin-13: central mediator of allergic asthma” Science; 282:2258-2261 (1998); the contents of each of which are herein incorporatedby reference in their entirety. Exemplary cells that may be usedaccording to these assays include Th2 cells. IL13, a Th2 type cytokine,is a potent stimulus for mucus production, airway hyper-responsivenessand allergic asthma. Th2 cells are a class of T cells that secrete IL4,IL10, IL13, IL5 and IL6. Factors that induce differentiation andactivation of Th2 cells play a major role in the initiation andpathogenesis of allergy and asthma. Primary T helper 2 cells aregenerated in in vitro culture under Th2 polarizing conditions usingperipheral blood lymphocytes isolated from cord blood. 124 HOSDO75 328Regulation of Caspase Apoptosis. Assays for caspase apoptosis are wellknown in the art and may be used or apoptosis in routinely modified toassess the ability of polypeptides of the invention (includingantibodies and pancreatic beta agonists or antagonists of the invention)to promote caspase protease-mediated apoptosis. cells. Apoptosis inpancreatic beta is associated with induction and progression ofdiabetes. Exemplary assays for caspase apoptosis that may be used orroutinely modified to test capase apoptosis activity of polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in: Loweth, AC, et al., FEBSLett, 400(3): 285-8 (1997); Saini, KS, et al., Biochem Mol Biol Int,39(6): 1229-36 (1996); Krautheim, A., et al., Br J Pharmacol, 129(4):687-94 (2000); Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 (2001);Suk K, et al., J Immunol, 166(7): 4481-9 (2001); Tejedo J, et al., FEBSLett, 459(2): 238-43 (1999); Zhang, S., et al., FEBS Lett, 455(3):315-20 (1999); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor etal., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, JAtheroscler Thromb 3(2): 75-80 (1996); the contents of each of which areherein incorporated by reference in its entirety. Pancreatic cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeRIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell linederived from a radiation induced transplantable rat islet cell tumor.The cells produce and secrete islet polypeptide hormones, and produceinsulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick etal. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad.Sci. 1980 77: 3519. 125 HOUDR07 329 Activation of Kinase assay. Kinaseassays, for examplek Elk-1 kinase assays, for ERK signal transductionthat Skeletal Muscle regulate cell proliferation or differentiation arewell known in the art and may be used or routinely Cell ERK modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Signalling antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Pathway Exemplary assays for ERK kinase activity that may be used orroutinely modified to test ERK kinase-induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Rat myoblastcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 126 HPEAD23 330 Stimulation of Assaysfor measuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 127 HPFCI36 331 Production of MIP-1alpha FMAT. Assays forimmunomodulatory proteins produced by activated dendritic cellsMIP1alpha that upregulate monocyte/macrophage and T cell chemotaxis arewell known in the art and may be used or routinely modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to mediate immunomodulation,modulate chemotaxis, and modulate T cell differentiation. Exemplaryassays that test for immunomodulatory proteins evaluate the productionof chemokines, such as macrophage inflammatory protein 1 alpha (MIP-1a),and the activation of monocytes/macrophages and T cells. Such assaysthat may be used or routinely modified to test immunomodulatory andchemotaxis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999);Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Satthaporn and Eremin, J R Coll Surg Ednb 45(1): 9-19 (2001);Drakes et al., Transp Immunol 8(1): 17-29 (2000); Verhasselt et al., JImmunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65:822-828 (1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 127 HPFCI36 331 Stimulation of Assays formeasuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 127 HPFCI36 331 Activation of Kinase assay. Kinase assays, forexample an GSK-3 kinase assay, for PI3 kinase signal Skeletal Mucletransduction that regulate glucose metabolism and cell survivial arewell-known in the art and may Cell PI3 Kinase be used or routinelymodified to assess the ability of polypeptides of the invention(including Signalling antibodies and agonists or antagonists of theinvention) to promote or inhibit glucose metabolism Pathway and cellsurvival. Exemplary assays for PI3 kinase activity that may be used orroutinely modified to test PI3 kinase-induced activity of polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271(2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), thecontents of each of which are herein incorporated by ref- erence in itsentirety. Rat myoblast cells that may be used according to these assaysare publicly available (e.g., through the ATCC). Exemplary rat myoblastcells that may be used according to these assays include L6 cells. L6 isan adherent rat myoblast cell line, isolated from primary cultures ofrat thigh muscle, that fuses to form multinucleated myotubes andstriated fibers after culture in differentiation media. 128 HPFDI37 332Activation of Assays for the activation of transcription through theGamma Interferon Activation Site (GAS) transcription response elementare well-known in the art and may be used or routinely modified toassess the through GAS ability of polypeptides of the invention(including antibodies and agonists or antagonists of the responseelement invention) to modulate gene expression (commonly via STATtranscription factors) involved in a in immune cells wide variety ofcell functions. Exemplary assays for transcription through the GASresponse (such as element that may be used or routinely modified to testGAS-response element activity of eosinophils). polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainenet al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol155(10): 4582-4587 (1995); the contents of each of which are hereinincorporated by reference in its entirety. Moreover, exemplary assaysthat may be used or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) to activate or inhibit activation ofimmune cells include assays disclosed and/or cited in: Mayumi M.,“EoL-1, a human eosinophilic cell line” Leuk Lymphoma; Jun; 7(3): 243-50(1992); Bhattacharya S, “Granulocyte macrophage colony-stimulatingfactor and interleukin-5 activate STAT5 and induce CIS1 mRNA in humanperipheral blood eosinophils” Am J Respir Cell Mol Biol; Mar; 24(3):312-6 (2001); and, Du J, et al., “Engagement of the CrkL adapter ininterleukin-5 signaling in eosinophils” J Biol Chem; Oct 20; 275(42):33167-75 (2000); the contents of each of which are herein incorporatedby reference in its entirety. Exemplary cells that may be used accordingto these assays include eosinophils. Eosinophils are a type of immunecell important in the late stage of allergic reactions; they arerecruited to tissues and mediate the inflammtory response of late stageallergic reaction. Increases in GAS mediated transcription ineosinophils is typically a result of STAT activation, normally a directconsequence of interleukin or other cytokine receptor stimulation (e.g.IL3, IL5 or GMCSF). 128 HPFDI37 332 Regulation of Caspase Apoptosis.Assays for caspase apoptosis are well known in the art and may be usedor apoptosis in routinely modified to assess the ability of polypeptidesof the invention (including antibodies and pancreatic beta agonists orantagonists of the invention) to promote caspase protease-mediatedapoptosis. cells. Apoptosis in pancreatic beta is associated withinduction and progression of diabetes. Exemplary assays for caspaseapoptosis that may be used or routinely modified to test capaseapoptosis activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in: Loweth, AC, et al., FEBS Lett, 400(3): 285-8(1997); Saini, KS, et al., Biochem Mol Biol Int, 39(6): 1229-36 (1996);Krautheim, A., et al., Br J Pharmacol, 129(4): 687-94 (2000); Chandra J,et al., Diabetes, 50 Suppl 1: S44-7 (2001); Suk K, et al., J Immunol,166(7): 4481-9 (2001); Tejedo J, et al., FEBS Lett, 459(2): 238-43(1999); Zhang, S., et al., FEBS Lett, 455(3): 315-20 (1999); Lee et al.,FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3):209-218 (2000); and Karsan and Harlan, J Atheroscler Tbromb 3(2): 75-80(1996); the contents of each of which are herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include RIN-m. RIN-m is a rat adherentpancreatic beta cell insulinoma cell line derived from a radiationinduced transplantable rat islet cell tumor. The cells produce andsecrete islet polypeptide hormones, and produce insulin, somatostatin,and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad.Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 129HPIAA80 333 Activation of Kinase assay. Kinase assays, for example anElk-1 kinase assay, for ERK signal transduction that Adipocyte ERKregulate cell proliferation or differentiation are well known in the artand may be used or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 129 HPIAA80 333 Regulationof Assays for the regulation of transcription of Malic Enzyme arewell-known in the art and may be transcription of used or routinelymodified to assess the ability of polypeptides of the invention(including Malic Enzyme in antibodies and agonists or antagonists of theinvention) to regulate transcription of Malic Enzyme, adipocytes a keyenzyme in lipogenesis. Malic enzyme is involved in lipogenesisand itsexpression is stimulted by insulin. ME promoter contains two directrepeat (DR1)- like elements MEp and MEd identified as putative PPARresponse elements. ME promoter may also responds to AP1 and othertranscription factors. Exemplary assays that may be used or routinelymodified to test for regulation of transcription of Malic Enzyme (inadipoocytes) by polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed in:Streeper, R. S., et al., Mol Endocrinol, 12(11): 1778-91 (1998);Garcia-Jimenez, C., et al., Mol Endocrinol, 8(10): 1361-9 (1994);Barroso, I., et al., J Biol Chem, 274(25): 17997-8004 (1999); Ijpenberg,A., et al., J Biol Chem, 272(32): 20108-20117 (1997); Berger, et al.,Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216:362-368 (1992), the contents of each of which is herein incorporated byreference in its entirety. Hepatocytes that may be used according tothese assays are publicly available (e.g., through the ATCC) and/or maybe routinely generated. Exemplary hepatocytes that may be used accordingto these assays includes the H4IIE rat liver hepatoma cell line. 130HPJCW58 334 Regulation of Assays for the regulation of transcriptionthrough the FAS promoter element are well-known in the transcription artand may be used or routinely modified to assess the ability ofpolypeptides of the invention through the FAS (including antibodies andagonists or antagonists of the invention) to activate the FAS promoterpromoter element element in a reporter construct and to regulatetranscription of FAS, a key enzyme for lipogenesis. in hepatocytes FASpromoter is regulated by many transcription factors including SREBP.Insulin increases FAS gene transcription in livers of diabetic mice.This stimulation of transcription is also somewhat glucose dependent.Exemplary assays that may be used or routinely modified to test for FASpromoter element activity (in hepatocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Xiong, S., et al., Proc Natl AcadSci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem,260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65(1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al.,Methods in Enzymol. 216: 362-368 (1992), the contents of each of whichis herein incorporated by reference in its entirety. Hepatocytes thatmay be used according to these assays, such as H4IIE cells, are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assays includerat liver hepatoma cell line(s) inducible with glucocorticoids, insulin,or cAMP derivatives. 131 HPRBH85 335 Stimulation of Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 132 HPRCA64 336 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 133 HPRCD35 337Stimulation of Assays for measuring calcium flux are well-known in theart and may be used or routinely Calcium Flux in modified to assess theability of polypeptides of the invention (including antibodies andagonists or pancreatic beta antagonists of the invention) to mobilizecalcium. For example, the FLPR assay may be used to cells. measureinflux of calcium. Cells normally have very low concentrations ofcytosolic calcium compared to much higher extracellular calcium.Extracellular factors can cause an influx of calcium, leading toactivation of calcium responsive signaling pathways and alterations incell functions. Exemplary assays that may be used or routinely modifiedto measure calcium flux by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Satin LS, et al., Endocrinology, 136(10): 4589-601 (1995);Mogami H, et al., Endocrinology, 136(7): 2960-6 (1995); Richardson SB,et al., Biochem J, 288 (Pt 3): 847-51 (1992); and, Meats, JE, et al.,Cell Calcium 1989 Nov-Dec; 10(8): 535-41 (1989), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 133 HPRCD35 337 IL-13 in HMC 134HPTRM02 338 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ohtani KI, etal., Endocrinology, 139(1): 172-8 (1998); Krautheim A, et al, Exp ClinEndocrinol Diabetes, 107 (1): 29-34 (1999), the contents of each ofwhich is herein incorporated by reference in its entirety. Pancreaticcells that may be used according to these assays are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays includeHITT15 Cells. HITT15 are an adherent epithelial cell line establishedfrom Syrian hamster islet cells transformed with SV40. These cellsexpress glucagon, somatostatin, and glucocorticoid receptors. The cellssecrete insulin, which is stimulated by glucose and glucagon andsuppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lordand Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl.Acad. Sci. USA 78: 4339-4343, 1981. 134 HPTRM02 338 Activation of Assaysfor the activation of transcription through the Gamma InterferonActivation Site (GAS) transcription response element are well-known inthe art and may be used or routinely modified to assess the through GASability of polypeptides of the invention (including antibodies andagonists or antagonists of the response element invention) to regulateSTAT transcription factors and modulate gene expression involved in awide in immune cells variety of cell functions. Exemplary assays fortranscription through the GAS response element that (such as T-cells).may be used or routinely modified to test GAS-response element activityof polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al.,J Immunol 155(10): 4582-4587 (1995), the contents of each of which areherein incorporated by reference in its entirety. Exemplary mouse Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the CTLL cell line, which is a suspensionculture of IL-2 dependent cytotoxic T cells. 134 HPTRM02 338 Activationof This reporter assay measures activation of the GATA-3 signalingpathway in HMC-1 human mast transcription cell line. Activation ofGATA-3 in mast cells has been linked to cytokine and chemokine throughGATA-3 production. Assays for the activation of transcription throughthe GATA3 response element are response element well-known in the artand may be used or routinely modified to assess the ability ofpolypeptides of in immune cells the invention (including antibodies andagonists or antagonists of the invention) to regulate GATA3 (such asmast transcription factors and modulate expression of mast cell genesimportant for immune response cells). development. Exemplary assays fortranscription through the GATA3 response element that may be used orroutinely modified to test GATA3-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999);Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zhengand Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol CellBiol 14(6): 4286-4294 (1994), the contents of each of which are hereinincorpor- ated by reference in its entirety. Mast cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary human mast cells that may be used acc- ording to theseassays include the HMC-1 cell line, which is an immature human mast cellline established from the peripheral blood of a patient with mast cellleukemia, and exhibits many characteristics of immature mast cells. 135HRAAD30 339 Activation of Kinase assay. Kinase assays, for example anElk-1 kinase assay, for ERK signal transduction that Adipocyte ERKregulate cell proliferation or differentiation are well known in the artand may be used or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 136 HRADF49 340 Activationof Kinase assay. Kinase assays, for example an Elk-1 kinase assay, forERK signal transduction that Adipocyte ERK regulate cell proliferationor differentiation are well known in the art and may be used orroutinely Signaling modified to assess the ability of polypeptides ofthe invention (including antibodies and agonists or Pathway antagonistsof the invention) to promote or inhibit cell proliferation, activation,and differentiation. Exemplary assays for ERK kinase activity that maybe used or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 136 HRADF49 340Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune cells transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe (such as T-cells). polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2):105-117 (1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension culture of T cells withcytotoxic activity. 137 HRDDQ39 341 Regulation of Assays for theregulation of transcription through the DMEF1 response element arewell-known in transcription via the art and may be used or routinelymodified to assess the ability of polypeptides of the invention DMEF1response (including antibodies and agonists or antagonists of theinvention) to activate the DMEF1 response element in element in areporter construct (such as that containing the GLUT4 promoter) and toregulate insulin adipocytes and production. The DMEF1 response elementis present in the GLUT4 promoter and binds to MEF2 pre-adipocytestranscription factor and another transcription factor that is requiredfor insulin regulation of Glut4 expression in skeletal muscle. GLUT4 isthe primary insulin-responsive glucose transporter in fat and muscletissue. Exemplary assays that may be used or routinely modified to testfor DMEF1 response element activity (in adipocytes and pre-adipocytes)by polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 137HRDDQ39 341 Activation of Assays for the activation of transcriptionthrough the Serum Response Element (SRE) are well- transcription knownin the art and may be used or routinely modified to assess the abilityof polypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune cells transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe (such as T-cells). polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2):105-117 (1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension culture of T cells withcytotoxic activity. 137 HRDDQ39 341 Stimulation of Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 138 HRDEX93 342 Regulation of Assays forthe regulation of transcription through the FAS promoter element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughthe FAS (including antibodies and agonists or antagonists of theinvention) to activate the FAS promoter promoter element element in areporter construct and to regulate transcription of FAS, a key enzymefor lipogenesis. in hepatocytes FAS promoter is regulated by manytranscription factors including SREBP. Insulin increases FAS genetranscription in livers of diabetic mice. This stimulation oftranscription is also somewhat glucose dependent. Exemplary assays thatmay be used or routinely modified to test for FAS promoter elementactivity (in hepatocytes) by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Xiong, S., et al., Proc Natl Acad Sci U.S.A., 97(8):3948-53 (2000); Roder, K., et al., Eur J Biochem, 260(3): 743-51 (1999);Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65 (1996); Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Hepatocytes that may be usedaccording to these assays, such as H4IIE cells, are publicly available(e.g., through the ATCC) and/or may be routinely generated. Exemplaryhepatocytes that may be used according to these assays include rat liverhepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMPderivatives. 139 HROEA08 343 Activation of Kinase assay. Kinase assays,for example an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 140 HRTAP63 344 Regulationof Assays for the regulation of viability and proliferation of cells invitro are well-known in the art and viability and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1):172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1):29-34 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 141 HSAVW42 345 Stimulation of Assays for measuring calcium fluxare well-known in the art and may be used or routinely Calcium Flux inmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or pancreatic beta antagonists of theinvention) to mobilize calcium. For example, the FLPR assay may be usedto cells. measure influx of calcium. Cells normally have very lowconcentrations of cytosolic calcium compared to much higherextracellular calcium. Extracellular factors can cause an influx ofcalcium, leading to activation of calcium responsive signaling pathwaysand alterations in cell functions. Exemplary assays that may be used orroutinely modified to measure calcium flux by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 142 HSAYC41 346 Stimulation of Assays for measuring secretion ofinsulin are well-known in the art and may be used or routinely insulinsecretion modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or from pancreaticantagonists of the invention) to stimulate insulin secretion. Forexample, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 143 HSLHX15 347 Upregulation of HLA-DRFMAT. MHC class II is essential for correct presentation of antigen toCD4+ T cells. HLA-DR and Deregulation of MHC class II has beenassociated with autoimmune diseases (e.g., diabetes, activation ofrheumatoid arthritis, systemic lupus erythematosis, and multiplesclerosis). Assays for T cells immunomodulatory proteins expressed onMHC class II expressing T cells and antigen presenting cells are wellknown in the art and may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate the activation ofT cells, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof MHC class II products, such as HLA-DR antigens, and the activation ofT cells. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222(1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itohet al., J Histochem Cytochem 40(11): 1675-1683, the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or otherwise known in the art. Human T cellsare primary human lymphocytes that mature in the thymus and express a TCell receptor and CD3, CD4, or CD8. These cells mediate humoral orcell-mediated immunity and may be preactivated to enhance responsivenessto immunomodulatory factors. 144 HSNBM34 348 Regulation of Assays forthe regulation of transcription through the DMEF1 response element arewell-known in transcription via the art and may be used or routinelymodified to assess the ability of polypeptides of the invention DMEF1response (including antibodies and agonists or antagonists of theinvention) to activate the DMEF1 response element in element in areporter construct (such as that containing the GLUT4 promoter) and toregulate insulin adipocytes and production. The DMEF1 response elementis present in the GLUT4 promoter and binds to MEF2 pre-adipocytestranscription factor and another transcription factor that is requiredfor insulin regulation of Glut4 expression in skeletal muscle. GLUT4 isthe primary insulin-responsive glucose transporter in fat and muscletissue. Exemplary assays that may be used or routinely modified to testfor DMEF1 response element activity (in adipocytes and pre-adipocytes)by polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 145HSSEF77 349 Activation of T- Kinase assay. JNK and p38 kinase assays forsignal transduction that regulate cell proliferation, Cell p38 or JNKactivation, or apoptosis are well known in the art and may be used orroutinely modified to assess Signaling the ability of polypeptides ofthe invention (including antibodies and agonists or antagonists of thePathway. invention) to promote or inhibit immune cell (e.g. T-cell)proliferation, activation, and apoptosis. Exemplary assays for JNK andp38 kinase activity that may be used or routinely modified to test JNKand p38 kinase-induced activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include the assays disclosed in Forrer et al., Biol Chem 379(8-9):1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504 (1999);Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4):479-500 (1999); the contents of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension-culture cell line withcytotoxic activity. 145 HSSEF77 349 Insulin Secretion Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Shimizu, H.,et al., Endocr J, 47(3): 261-9 (2000); Salapatek, A. M., et al., MolEndocrinol, 13(8): 1305-17 (1999); Filipsson, K., et al., Ann N Y AcadSci, 865: 441-4 (1998); Olson, L. K., et al., J Biol Chem, 271(28):16544-52 (1996); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 145 HSSEF77 349 Production of Assays for measuring expression ofVCAM are well-known in the art and may be used or routinely VCAM inmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or endothelial cells antagonists ofthe invention) to regulate VCAM expression. For example, FMAT may beused to (such as human meaure the upregulation of cell surface VCAM-1expresssion in endothelial cells. Endothelial cells umbilical vein arecells that line blood vessels, and are involved in functions thatinclude, but are not limited to, endothelial cells angiogenesis,vascular permeability, vascular tone, and immune cell extravasation.Exemplary (HUVEC)) endothelial cells that may be used according to theseassays include human umbilical vein endothelial cells (HUVEC), which areavailable from commercial sources. The expression of VCAM (CD106), amembrane-associated protein, can be upregulated by cytokines or otherfactors, and contributes to the extravasation of lymphocytes, leucocytesand other immune cells from blood vessels; thus VCAM expression plays arole in promoting immune and inflammatory responses. 146 HT4FV41 350Stimulation of Assays for measuring secretion of insulin are well-knownin the art and may be used or routinely insulin secretion modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or from pancreatic antagonists of the invention)to stimulate insulin secretion. For example, insulin secretion is betacells. measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ahren, B., etal., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al.,Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett,377(2): 237-9 (1995); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 147 HT5FX79 351 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 148 HT5GR59 352Activation of Assays for the activation of transcription through the AP1response element are known in the art and transcription may be used orroutinely modified to assess the ability of polypeptides of theinvention (including through AP1 antibodies and agonists or antagonistsof the invention) to modulate growth and other cell functions. responseelement Exemplary assays for transcription through the AP1 responseelement that may be used or routinely in immune cells modified to testAP1-response element activity of polypeptides of the invention(including (such as T-cells). antibodies and agonists or antagonists ofthe invention) include assays disclosed in Berger et al., Gene 66: 1-10(1988); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Rellahanet al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al., Mol CellBiol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol 29(3):838-844 (1999), the contents of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension-culture cell line withcytotoxic activity. 148 HT5GR59 352 Stimulation of Assays for measuringsecretion of insulin are well-known in the art and may be used orroutinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 149 HTAE178 353 Upregulation of HLA-DRFMAT. MHC class II is essential for correct presentation of antigen toCD4+ T cells. HLA-DR and Deregulation of MHC class II has beenassociated with autoimmune diseases (e.g., diabetes, activation ofrheumatoid arthritis, systemic lupus erythematosis, and multiplesclerosis). Assays for T cells immunomodulatory proteins expressed onMHC class II expressing T cells and antigen presenting cells are wellknown in the art and may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to modulate the activation ofT cells, and/or mediate humoral or cell-mediated immunity. Exemplaryassays that test for immunomodulatory proteins evaluate the upregulationof MHC class II products, such as HLA-DR antigens, and the activation ofT cells. Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include, forexample, the assays disclosed in Miraglia et al., J BiomolecularScreening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practicalapproach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222(1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itohet al., J Histochem Cytochem 40(11): 1675-1683, the contents of each ofwhich are herein incorporated by reference in its entirety. Human Tcells that may be used according to these assays may be isolated usingtechniques disclosed herein or other- wise known in the art. Human Tcells are primary human lymphocytes that mature in the thymus andexpress a T Cell receptor and CD3, CD4, or CD8. These cells mediatehumoral cell-mediated immunity and may be preactivated to enhanceresponsiveness to immunomodulatory factors. 150 HTEAG62 354 Activationof Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3kinase signal transduction that Adipocyte PI3 regulate glucosemetabolism and cell survival are well-known in the art and may be usedor Kinase Signalling routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and Pathway agonistsor antagonists of the invention) to promote or inhibit glucosemetabolism and cell survival. Exemplary assays for PI3 kinase activitythat may be used or routinely modified to test PI3 kinase- inducedactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inForrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al.,Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8):1662-1666 (1999), the contents of each of which are herein incorporatedby reference in its entirety. Mouse adipocyte cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse adipocyte cells that may be used according tothese assays include 3T3- L1 cells. 3T3-L1 is an adherent mousepreadipocyte cell line that is a continous substrain of 3T3 fibroblastcells developed through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 150 HTEAG62 354 Regulation of Assays for theregulation of transcription of Malic Enzyme are well-known in the artand may be transcription of used or routinely modified to assess theability of polypeptides of the invention (including Malic Enzyme inantibodies and agonists or antagonists of the invention) to regulatetranscription of Malic Enzyme, adipocytes a key enzyme in lipogenesis.Malic enzyme is involved in lipogenesisand its expression is stimultedby insulin. ME promoter contains two direct repeat (DR1)- like elementsMEp and MEd identified as putative PPAR response elements. ME promotermay also responds to AP1 and other transcription factors. Exemplaryassays that may be used or routinely modified to test for regulation oftranscription of Malic Enzyme (in adipoocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Streeper, R. S., et al., MolEndocrinol, 12(11): 1778-91 (1998); Garcia-Jimenez, C., et al., MolEndocrinol, 8(10): 1361-9 (1994); Barroso, I., et al., J Biol Chem,274(25): 17997-8004 (1999); Ijpenberg, A., et al., J Biol Chem, 272(32):20108-20117 (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen,B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents ofeach of which is herein incorporated by reference in its entirety.Hepatocytes that may be used according to these assays are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assaysincludes the H4IIE rat liver hepatoma cell line. 151 HTEDJ28 355Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 151 HTEDJ28 355 Activationof Assays for the activation of transcription through the Nuclear Factorof Activated T cells (NFAT) transcription response element arewell-known in the art and may be used or routinely modified to assessthe through NFAT ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the response elementinvention) to regulate NFAT transcription factors and modulateexpression of genes involved in in immune cells immunomodulatoryfunctions. Exemplary assays for transcription through the NFAT response(such as natural element that may be used or routinely modified to testNFAT-response element activity of killer cells). polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Aramburuet al., J Exp Med 182(3): 801-810 (1995); De Boer et al., Int J BiochemCell Biol 31(10): 1221-1236 (1999); Fraser et al., Eur J Immunol 29(3):838-844 (1999); and Yeseen et al., J Biol Chem 268(19): 14285-14293(1993), the contents of each of which are herein incorporated byreference in its entirety. NK cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary humanNK cells that may be used according to these assays include the NK-YTcell line, which is a human natural killer cell line with cytolytic andcytotoxic activity. 152 HTEDS12 356 Activation of Kinase assay. Kinaseassays, for example an Elk-1 kinase assay, for ERK signal transductionthat Adipocyte ERK regulate cell proliferation or differentiation arewell known in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110(1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 153 HTEHA56 357 Activationof Kinase assay. Kinase assays, for example an GSK-3 assays, for PI3kinase signal transduction that Adipocyte PI3 regulate glucosemetabolism and cell survival are well-known in the art and may be usedor Kinase Signalling routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and Pathway agonistsor antagonists of the invention) to promote or inhibit glucosemetabolism and cell survival. Exemplary assays for PI3 kinase activitythat may be used or routinely modified to test PI3 kinase- inducedactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inForrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al.,Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8):1662-1666 (1999), the contents of each of which are herein incorporatedby reference in its entirety. Mouse adipocyte cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse adipocyte cells that may be used according tothese assays include 3T3- L1 cells. 3T3-L1 is an adherent mousepreadipocyte cell line that is a continous substrain of 3T3 fibroblastcells developed through clonal isolation and undergo a pre-adipocyte toadipose-like conversion under appropriate differentiation conditionsknown in the art. 153 HTEHA56 357 Activation of Kinase assay. Kinaseassays, for example an Elk-1 kinase assay, for ERK signal transductionthat Adipocyte ERK regulate cell proliferation or differentiation arewell known in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 154 HTEJD29 358 Stimulationof Assays for measuring calcium flux are well-known in the art and maybe used or routinely Calcium Flux in modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orpancreatic beta antagonists of the invention) to mobilize calcium. Forexample, the FLPR assay may be used to cells. measure influx of calcium.Cells normally have very low concentrations of cytosolic calciumcompared to much higher extracellular calcium. Extracellular factors cancause an influx of calcium, leading to activation of calcium responsivesignaling pathways and alterations in cell functions. Exemplary assaysthat may be used or routinely modified to measure calcium flux bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Satin LS, etal., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al.,Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J,288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 155 HTENR63 359 Regulation of Assays for the regulation oftranscription through the PEPCK promoter are well-known in the arttranscription and may be used or routinely modified to assess theability of polypeptides of the invention through the (includingantibodies and agonists or antagonists of the invention) to activate thePEPCK promoter PEPCK promoter in a reporter construct and regulate livergluconeogenesis. Exemplary assays for regulation of in hepatocytestranscription through the PEPCK promoter that may be used or routinelymodified to test for PEPCK promoter activity (in hepatocytes) ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Lochhead et al., Diabetes 49(6): 896-903 (2000); and Yeagley et al., JBiol Chem 275(23): 17814-17820 (2000), the contents of each of which isherein incorporated by reference in its entirety. Hepatocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary liverhepatoma cells that may be used according to these assays include H4llecells, which contain a tyrosine amino transferase that is inducible withglucocorticoids, insulin, or cAMP derivatives. 156 HTGGM44 360Regulation of Assays for the regulation of viability and proliferationof cells in vitro are well-known in the art and viability and may beused or routinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 157 HTHBZ06 361 Stimulation of Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 158 HTLBT80 362 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 158 HTLBT80 362Activation of Assays for the activation of transcription through the EGRresponse element are well-known in the transcription art and may be usedor routinely modified to assess the ability of polypeptides of theinvention through the EGR (including antibodies and agonists orantagonists of the invention) to regulate EGR transcription (EarlyGrowth factors and modulate expression of immunomodulatory genes.Exemplary assays for transcription Response) through the EGR responseelement that may be used or routinely modified to test EGR responseelement in element activity of polypeptides of the invention (includingantibodies and agonists or antagonists of immune cells the invention)include assays disclosed in: Richards JD, et al., J Immunol, 166(6):3855-3864 (2001); (such as B-cells). Dinkel, A, et al., J Exp Med,188(12): 2215-2224 (1998); and, Newton, JS, et al., Eur J Immunol 1996Apr; 26(4): 811-816 (1996), the contents of each of which are hereinincorporated by reference in its entirety. Immune cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary epithelial cells that may be used according to theseassays include the Raji cell line. 158 HTLBT80 362 Activation of Assaysfor the activation of transcription through the EGR response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughthe EGR (including antibodies and agonists or antagonists of theinvention) to regulate EGR transcription (Early Growth factors andmodulate expression of immunomodulatory genes. Exemplary assays fortranscription Response) through the EGR response element that may beused or routinely modified to test EGR response element in elementactivity of polypeptides of the invention (including antibodies andagonists or antagonists immune cells of the invention) include assaysdisclosed in: Richards JD, et al., J Immunol, 166(6): 3855-3864 (such asB-cells). (2001); Dinkel, A, et al., J Exp Med, 188(12): 2215-2224(1998); and, Newton, JS, et al., Eur J Immunol 1996 Apr; 26(4): 811-816(1996), the contents of each of which are herein incorporated byreference in its entirety. Immune cells that may be used according tothese assays are publicly available (e.g., through the ATCC). Exemplaryepithelial cells that may be used according to these assays include theRaji cell line. 158 HTLBT80 362 Activation of Assays for the activationof transcription through the NFKB response element are well-known intranscription the art and may be used or routinely modified to assessthe ability of polypeptides of the invention through NFKB (includingantibodies and agonists or antagonists of the invention) to regulateNFKB transcription response element factors and modulate expression ofimmunomodulatory genes. Exemplary assays for transcription in immunecells through the NFKB response element that may be used or rountinelymodified to test NFKB- (such as B-cells). response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Gri G, etal., Biol Chem, 273(11): 6431-6438 (1998); Pyatt DW, et al., Cell BiolToxicol 2000; 16(1): 41-51 (2000); Berger et al., Gene 66: 1-10 (1998);Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn etal., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al,Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3):801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contentsof each of which are herein incorporated by reference in its entirety.Immune cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary immune cells that may beused according to these assays include the Reh B-cell line. 159 HTLDU78363 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: FriedrichsenBN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al.,Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998Jul 10; 273(28): 17771-9 (1998), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 159 HTLDU78 363 Activation of T- Kinaseassay. JNK and p38 kinase assays for signal transduction that regulatecell proliferation, Cell p38 or JNK activation, or apoptosis are wellknown in the art and may be used or routinely modified to assessSignaling the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the Pathway. invention) topromote or inhibit immune cell (e.g. T-cell) proliferation, activation,and apoptosis. Exemplary assays for JNK and p38 kinase activity that maybe used or routinely modified to test JNK and p38 kinase-inducedactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include the assays disclosedin Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Gupta et al.,Exp Cell Res 247(2): 495-504 (1999); Kyriakis JM, Biochem Soc Symp 64:29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and CobbMH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension-culture cell line with cytotoxic activity. 159 HTLDU78 363Production of IL-6 FMAT. IL-6 is produced by T cells and has strongeffects on B cells. IL-6 participates in IL-4 IL-6 induced IgEproduction and increases IgA production (IgA plays a role in mucosalimmunity). IL-6 induces cytotoxic T cells. Deregulated expression ofIL-6 has been linked to autoimmune disease, plasmacytomas, myelomas, andchronic hyperproliferative diseases. Assays for immunomodulatory anddifferentiation factor proteins produced by a large variety of cellswhere the expression level is strongly regulated by cytokines, growthfactors, and hormones are well known in the art and may be used orroutinely modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) to mediate immunomodulation and differentiation and modulateT cell proliferation and function. Exemplary assays that test forimmunomodulatory proteins evaluate the production of cytokines, such asIL-6, and the stimulation and upregulation of T cell proliferation andfunctional activities. Such assays that may be used or routinelymodified to test immunomodulatory and dififerentiation activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Miraglia etal., J Biomolecular Screening 4: 193-204(1999); Rowland et al.,“Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); andVerhasselt et al., J Immunol 158: 2919-2925 (1997), the contents of eachof which are herein incorporated by reference in its entirety. Humandendritic cells that may be used according to these assays may beisolated using techniques disclosed herein or otherwise known in theart. Human dendritic cells are antigen presenting cells in suspensionculture, which, when activated by antigen and/or cytokines, initiate andupregulate T cell proliferation and functional activities. 160 HTLFA13364 Regulation of Assays for the regulation of viability andproliferation of cells in vitro are well-known in the art and viabilityand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including proliferation of antibodies andagonists or antagonists of the invention) to regulate viability andproliferation of pancreatic beta pancreatic beta cells. For example, theCell Titer-Glo luminescent cell viability assay measures the cells.number of viable cells in culture based on quantitation of the ATPpresent which signals the presence of metabolically active cells.Exemplary assays that may be used or routinely modified to testregulation of viability and proliferation of pancreatic beta cells bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: FriedrichsenBN, et al., Mol Endocrinol, 15(1): 136-48 (2001); Huotari MA, et al.,Endocrinology, 139(4): 1494-9 (1998); Hugl SR, et al., J Biol Chem 1998Jul 10; 273(28): 17771-9 (1998), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 161 HTLGI89 365 Stimulation of Assays formeasuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 162 HTNBK13 366 Regulation of Assays for the regulation ofviability and proliferation of cells in vitro are well-known in the artviability and and may be used or routinely modified to assess theability of polypeptides of the invention (includ- proliferation of ingantibodies and agonists or antagonists of the invention) to regulateviability and proliferation pancreatic beta of pancreatic beta cells.For example, the Cell Titer-Glo luminescent cell viability assaymeasures cells. the number of viable cells in culture based onquantitation of the ATP present which signals the presence ofmetabolically active cells. Exemplary assays that may be used orroutinely mod- ified to test regulation of viability and proliferationof pancreatic beta cells by polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in: Friedrichsen BN, et al., Mol Endocrinol, 15(1): 136-48(2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9 (1998); HuglSR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771- 9 (1998), thecontents of each of which is herein incorporated by reference in itsentirety. Pancreatic cells that may be used according to these assaysare publicly available (e.g., through the ATCC) and/ or may be routinelygenerated. Exemplary pancreatic cells that may be used according tothese assays include rat INS-1 cells. INS-1 cells are a semi-adherentcell line established from cells isolated from an X-ray induced rattransplantable insulinoma. These cells retain characteristics typical ofnative pancreatic beta cells including glucose inducible insulinsecretion. References: Asfari et al. Endocrinology 1992 130: 167. 162HTNBK13 366 Production of TNFa FMAT. Assays for immunomodulatoryproteins produced by activated macrophages, T cells, TNF alpha byfibroblasts, smooth muscle, and other cell types that exert a widevariety of inflammatory and dendritic cells cytotoxic effects on avariety of cells are well known in the art and may be used or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and agonists or antagonists of the invention) tomediate immunomodulation, modulate inflammation and cytotoxicity.Exemplary assays that test for immunomodulatory proteins evaluate theproduction of cytokines such as tumor necrosis factor alpha (TNFa), andthe induction or inhibition of an inflammatory or cytotoxic response.Such assays that may be used or routinely modified to testimmunomodulatory activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999);Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160(2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198);Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., JImmunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65:822-828 (1999), the contents of each of which are herein incorporated byreference in its entirety. Human dendritic cells that may be usedaccording to these assays may be isolated using techniques disclosedherein or otherwise known in the art. Human dendritic cells are antigenpresenting cells in suspension culture, which, when activated by antigenand/or cytokines, initiate and upregulate T cell proliferation andfunctional activities. 162 HTNBK13 366 Production of Assays forproduction of IL-10 and activation of T-cells are well known in the artand may be used IL-10 and or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and activation ofagonists or antagonists of the invention) to stimulate or inhibitproduction of IL-10 and/or activation T-cells. of T-cells. Exemplaryassays that may be used or routinely modified to assess the ability ofpolypeptides and antibodies of the invention (including agonists orantagonists of the invention) to modulate IL-10 production and/or T-cellproliferation include, for example, assays such as disclosed and/orcited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” BrMed Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2cell-directed therapy for asthma” Pharmacology & Therapeutics; 88:187-196 (2000); the contents of each of which are herein incorporated byreference in their entirety. Exemplary cells that may be used accordingto these assays include Th2 cells. IL10 secreted from Th2 cells may bemeasured as a marker of Th2 cell activation. Th2 cells are a class of Tcells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that inducedifferentiation and activation of Th2 cells play a major role in theinitiation and pathogenesis of allergy and asthma. Primary T helper 2cells are generated via in vitro culture under Th2 polarizing conditionsusing peripheral blood lymphocytes isolated from cord blood. 163 HTOAM11367 Myoblast cell Assays for muscle cell proliferation are well known inthe art and may be used or routinely proliferation modified to assessthe ability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to stimulate or inhibitmyoblast cell proliferation. Exemplary assays for myoblast cellproliferation that may be used or routinely modified to test activity ofpolypeptides and antibodies of the invention (including agonists orantagonists of the invention) include, for example, assays disclosed in:Soeta, C., et al. “Possible role for the c-ski gene in the proliferationof myogenic cells in regenerating skeletal muscles of rats” Dev GrowthDiffer Apr; 43(2): 155-64 (2001); Ewton DZ, et al., “IGF bindingproteins-4, -5 and -6 may play specialized roles during L6 myoblastproliferation and differentiation” J Endocrinol Mar; 144(3): 539-53(1995); and, Pampusch MS, et al., “Effect of transforming growth factorbeta on proliferation of L6 and embryonic porcine myogenic cells” J CellPhysiol Jun; 143(3): 524-8 (1990); the contents of each of which areherein incorporated by reference in their entirety. Exemplary myoblastcells that may be used according to these assays include the ratmyoblast L6 cell line. Rat myoblast L6 cells are an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuse to form multinucleated myotubes and striated fibers afterculture in differentiation media. 163 HTOAM11 367 Activation of Assaysfor the activation of transcription through the AP1 response element areknown in the art transcription and may be used or routinely modified toassess the ability of polypeptides of the invention through AP1(including antibodies and agonists or antagonists of the invention) tomodulate growth and other response element cell functions. Exemplaryassays for transcription through the AP1 response element that may be inimmune cells used or routinely modified to test AP1-response elementactivity of polypeptides of the invention (such as T-cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1988); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Rellahan et al., J Biol Chem 272(49):30806-30811 (1997); Chang et al., Mol Cell Biol 18(9): 4986-4993 (1998);and Fraser et al., Eur J Immunol 29(3): 838-844 (1999), the contents ofeach of which are herein incorporated by reference in its entirety. Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary mouse T cells that may be usedaccording to these assays include the CTLL cell line, which is an IL-2dependent suspension-culture cell line with cytotoxic activity. 163HTOAM11 367 Activation of Assays for the activation of transcriptionthrough the cAMP response element are well-known in the transcriptionart and may be used or routinely modified to assess the ability ofpolypeptides of the invention through cAMP (including antibodies andagonists or antagonists of the invention) to increase cAMP and regulateresponse element CREB transcription factors, and modulate expression ofgenes involved in a wide variety of cell in immune cells functions.Exemplary assays for transcription through the cAMP response elementthat may be used (such as T-cells). or routinely modified to testcAMP-response element activity of polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullenand Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., ProcNatl Acad Sci USA 85: 6342-6346 (1988); Black et al., Virus Genes 15(2):105-117 (1997); and Belkowski et al., J Immunol 161(2): 659-665 (1998),the contents of each of which are herein incorporated by reference inits entirety. T cells that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary mouse T cellsthat may be used according to these assays include the CTLL cell line,which is a suspension culture of IL-2 dependent cytotoxic T cells. 163HTOAM11 367 Activation of Assays for the activation of transcriptionthrough the Gamma Interferon Activation Site (GAS) transcriptionresponse element are well-known in the art and may be used or routinelymodified to assess the through GAS ability of polypeptides of theinvention (including antibodies and agonists or antagonists of theresponse element invention) to regulate STAT transcription factors andmodulate gene expression involved in a wide in immune cells variety ofcell functions. Exemplary assays for transcription through the GASresponse element that (such as T-cells). may be used or routinelymodified to test GAS-response element activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainenet al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol155(10): 4582-4587 (1995), the contents of each of which are hereinincorporated by reference in its entirety. Exemplary mouse T cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary T cells that may be used according to theseassays include the CTLL cell line, which is a suspension culture of IL-2dependent cytotoxic T cells. 163 HTOAM11 367 Activation of Assays forthe activation of transcription through the Serum Response Element (SRE)are well- transcription known in the art and may be used or routinelymodified to assess the ability of polypeptides of the through seruminvention (including antibodies and agonists or antagonists of theinvention) to regulate the serum response element response factors andmodulate the expression of genes involved in growth. Exemplary assaysfor in immune cells transcription through the SRE that may be used orroutinely modified to test SRE activity of the (such as T-cells).polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);and Black et al., Virus Genes 12(2): 105-117 (1997), the content of eachof which are herein incorporated by reference in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse T cells that may be used according tothese assays include the CTLL cell line, which is an IL-2 dependentsuspension culture of T cells with cytotoxic activity. 163 HTOAM11 367Stimulation of Assays for measuring secretion of insulin are well-knownin the art and may be used or routinely insulin secretion modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or from pancreatic antagonists of the invention)to stimulate insulin secretion. For example, insulin secretion is betacells. measured by FMAT using anti-rat insulin antibodies. Insulinsecretion from pancreatic beta cells is upregulated by glucose and alsoby certain proteins/peptides, and disregulation is a key component indiabetes. Exemplary assays that may be used or routinely modified totest for stimulation of insulin secretion (from pancreatic cells) bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Ahren, B., etal., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al.,Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett,377(2): 237-9 (1995); and, Miraglia S et. al., Journal of BiomolecularScreening, 4: 193-204 (1999), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include rat INS-1 cells. INS-1cells are a semi-adherent cell line established from cells isolated froman X-ray induced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 163 HTOAM11 367 Activation of Assays forthe activation of transcription through the CD28 response element arewell-known in the transcription art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughCD28 (including antibodies and agonists or antagonists of the invention)to stimulate IL-2 expression in response element T cells. Exemplaryassays for transcription through the CD28 response element that may beused or in immune cells routinely modified to test CD28-response elementactivity of polypeptides of the invention (such as T-cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); McGuire and Iacobelli, J Immunol 159(3):1319-1327 (1997); Parra et al., J Immunol 166(4): 2437-2443 (2001); andButscher et al., J Biol Chem 3(1): 552-560 (1998), the contents of eachof which are herein incorporated by ref- erence in its entirety. T cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary human T cells that may be used according tothese assays include the JURKAT cell line, which is a suspension cultureof leukemia cells that produce IL-2 when stimulated. 163 HTOAM11 367Production of IFNgamma FMAT. IFNg plays a central role in the immunesystem and is considered to be a IFNgamma using proinflammatorycytokine. IFNg promotes TH1 and inhibits TH2 differentiation; promotesIgG2a a T cells and inhibits IgE secretion; induces macrophageactivation; and increases MHC expression. Assays for immunomodulatoryproteins produced by T cells and NK cells that regulate a variety ofinflammatory activities and inhibit TH2 helper cell functions are wellknown in the art and may be used or routinely modified to assess theability of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) to mediate immunomodulation,regulate inflammatory activities, modulate TH2 helper cell function,and/or mediate humoral or cell- mediated immunity. Exemplary assays thattest for immunomodulatory proteins evaluate the production of cytokines,such as Interferon gamma (IFNg), and the activation of T cells. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include the assays disclosedin Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowlandet al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000);Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al.,Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15:749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), thecontents of each of which are herein incorporated by reference in itsentirety. Human T cells that may be used according to these assays maybe isolated using techniques disclosed herein or otherwise known in theart. Human T cells are primary human lymphocytes that mature in thethymus and express a T Cell receptor and CD3, CD4, or CD8. These cellsmediate humoral or cell-mediated immunity and may be preactivated toenhance responsiveness to immunomodulatory factors. 164 HTOHO21 368Activation of Kinase assay. Kinase assays, for example an Elk-1 kinaseassay, for ERK signal transduction that Adipocyte ERK regulate cellproliferation or differentiation are well known in the art and may beused or routinely Signaling modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orPathway antagonists of the invention) to promote or inhibit cellproliferation, activation, and differentiation. Exemplary assays for ERKkinase activity that may be used or routinely modified to test ERKkinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include theassays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110(1998);Le Marchand-Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Mouse adipocyte cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary mouse adipocyte cells that may be usedaccording to these assays include 3T3-L1 cells. 3T3-L1 is an adherentmouse preadipocyte cell line that is a continuous substrain of 3T3fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 164 HTOHO21 368 Activationof Kinase assay. Kinase assays, for example an GSK-3 kinase assay, forPI3 kinase signal Skeletal Mucle transduction that regulate glucosemetabolism and cell survivial are well-known in the art and may Cell PI3Kinase be used or routinely modified to assess the ability ofpolypeptides of the invention (including Signalling antibodies andagonists or antagonists of the invention) to promote or inhibit glucosemetabolism Pathway and cell survival. Exemplary assays for PI3 kinaseactivity that may be used or routinely modified to test PI3kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al.,Diabetes 48(8): 1662-1666 (1999), the contents of each of which areherein incorporated by ref- erence in its entirety. Rat myoblast cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 165 HTPCO75 369 Regulation of Assaysfor the regulation of viability and proliferation of cells in vitro arewell-known in the art and viability and may be used or routinelymodified to assess the ability of polypeptides of the invention(including proliferation of antibodies and agonists or antagonists ofthe invention) to regulate viability and proliferation of pancreaticbeta pancreatic beta cells. For example, the Cell Titer-Glo luminescentcell viability assay measures the cells. number of viable cells inculture based on quantitation of the ATP present which signals thepresence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ohtani KI, et al., Endocrinology, 139(1):172-8 (1998); Krautheim A, et al, Exp Clin Endocrinol Diabetes, 107 (1):29-34 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 166 HTSFJ32 370 Activation of Assays for the activation oftranscription through the Serum Response Element (SRE) are well-transcription known in the art and may be used or routinely modified toassess the ability of polypeptides of the through serum invention(including antibodies and agonists or antagonists of the invention) toregulate the serum response element response factors and modulate theexpression of genes involved in growth. Exemplary assays for in immunecells transcription through the SRE that may be used or routinelymodified to test SRE activity of the (such as T-cells). polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Berger et al., Gene 66: 1-10(1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992);Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); and Blacket al., Virus Genes 12(2): 105-117 (1997), the content of each of whichare herein incorporated by reference in its entirety. T cells that maybe used according to these assays are publicly available (e.g., throughthe ATCC). Exemplary mouse T cells that may be used according to theseassays include the CTLL cell line, which is an IL-2 dependent suspensionculture of T cells with cytotoxic activity. 166 HTSFJ32 370 Activationof Kinase assay. Kinase assays, for example an GSK-3 kinase assay, forPI3 kinase signal Skeletal Mucle transduction that regulate glucosemetabolism and cell survivial are well-known in the art and may Cell PI3Kinase be used or routinely modified to assess the ability ofpolypeptides of the invention (including Signalling antibodies andagonists or antagonists of the invention) to promote or inhibit glucosemetabolism Pathway and cell survival. Exemplary assays for PI3 kinaseactivity that may be used or routinely modified to test PI3kinase-induced activity of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998);Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al.,Diabetes 48(8): 1662-1666 (1999), the contents of each of which areherein incorporated by ref- erence in its entirety. Rat myoblast cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 167 HTTCB60 371 Stimulation of Assaysfor measuring secretion of insulin are well-known in the art and may beused or routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 168 HTTEE41 372 Stimulation of Assays formeasuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 168 HTTEE41 372 Production of Assays for production of IL-13 andactivation of T-cells are well known in the art and may be used IL-13and or routinely modified to assess the ability of polypeptides of theinvention (including antibodies and activation of agonists orantagonists of the invention) to stimulate or inhibit production ofIL-13 and/or activation T-cells. of T-cells. Exemplary assays for IL-13production that may be used or routinely modified to test activity ofpolypeptides and antibodies of the invention (including agonists orantagonists of the invention) include, for example, assays such asdisclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13independently of IL-4 in Experimental asthma” Science; 282: 2261-2263(1998), and Wills-Karp M, et al., “Interleukin-13: central mediator ofallergic asthma” Science; 282: 2258-2261 (1998); the contents of each ofwhich are herein incorporated by reference in their entirety. Exemplarycells that may be used according to these assays include Th2 cells.IL13, a Th2 type cytokine, is a potent stimulus for mucus production,airway hyper-responsiveness and allergic asthma. Th2 cells are a classof T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors thatinduce differentiation and activation of Th2 cells play a major role inthe initiation and pathogenesis of allergy and asthma. Primary T helper2 cells are generated in in vitro culture under Th2 polarizingconditions using peripheral blood lymphocytes isolated from cord blood.169 HTWEH94 373 Stimulation of Assays for measuring secretion of insulinare well-known in the art and may be used or routinely insulin secretionmodified to assess the ability of polypeptides of the invention(including antibodies and agonists or from pancreatic antagonists of theinvention) to stimulate insulin secretion. For example, insulinsecretion is beta cells. measured by FMAT using anti-rat insulinantibodies. Insulin secretion from pancreatic beta cells is upregulatedby glucose and also by certain proteins/peptides, and disregulation is akey component in diabetes. Exemplary assays that may be used orroutinely modified to test for stimulation of insulin secretion (frompancreatic cells) by polypeptides of the invention (including antibodiesand agonists or antagonists of the invention) include assays disclosedin: Ahren, B., et al., Am J Physiol, 277(4 Pt 2): R959-66 (1999); Li,M., et al., Endocrinology, 138(9): 3735-40 (1997); Kim, K. H., et al.,FEBS Lett, 377(2): 237-9 (1995); and, Miraglia S et. al., Journal ofBiomolecular Screening, 4: 193-204 (1999), the contents of each of whichis herein incorporated by reference in its entirety. Pancreatic cellsthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplarypancreatic cells that may be used according to these assays include ratINS-1 cells. INS-1 cells are a semi-adherent cell line established fromcells isolated from an X-ray induced rat transplantable insulinoma.These cells retain characteristics typical of native pancreatic betacells including glucose inducible insulin secretion. References: Asfariet al. Endocrinology 1992 130: 167. 170 HTXFA72 374 Stimulation ofAssays for measuring calcium flux are well-known in the art and may beused or routinely Calcium Flux in modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orpancreatic beta antagonists of the invention) to mobilize calcium. Forexample, the FLPR assay may be used to cells. measure influx of calcium.Cells normally have very low concentrations of cytosolic calciumcompared to much higher extracellular calcium. Extracellular factors cancause an influx of calcium, leading to activation of calcium responsivesignaling pathways and alterations in cell functions. Exemplary assaysthat may be used or routinely modified to measure calcium flux bypolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in: Satin LS, etal., Endocrinology, 136(10): 4589-601 (1995); Mogami H, et al.,Endocrinology, 136(7): 2960-6 (1995); Richardson SB, et al., Biochem J,288 (Pt 3): 847-51 (1992); and, Meats, JE, et al., Cell Calcium 1989Nov-Dec; 10(8): 535-41 (1989), the contents of each of which is hereinincorporated by reference in its entirety. Pancreatic cells that may beused according to these assays are publicly available (e.g., through theATCC) and/or may be routinely generated. Exemplary pancreatic cells thatmay be used according to these assays include HITT15 Cells. HITT15 arean adherent epithelial cell line established from Syrian hamster isletcells transformed with SV40. These cells express glucagon, somatostatin,and glucocorticoid receptors. The cells secrete insulin, which isstimulated by glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 171 HTXKF95 375 Activation of Kinase assay. Kinase assays, forexamplek Elk-1 kinase assays, for ERK signal transduction that SkeletalMuscle regulate cell proliferation or differentiation are well known inthe art and may be used or routinely Cell ERK modified to assess theability of polypeptides of the invention (including antibodies andagonists or Signalling antagonists of the invention) to promote orinhibit cell proliferation, activation, and differentiation. PathwayExemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Rat myoblastcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary rat myoblast cells that may be usedaccording to these assays include L6 cells. L6 is an adherent ratmyoblast cell line, isolated from primary cultures of rat thigh muscle,that fuses to form multinucleated myotubes and striated fibers afterculture in differentiation media. 172 HUKDF20 376 Regulation of Assaysfor the regulation of transcription through the DMEF1 response elementare well-known in transcription via the art and may be used or routinelymodified to assess the ability of polypeptides of the invention DMEF1response (including antibodies and agonists or antagonists of theinvention) to activate the DMEF1 response element in element in areporter construct (such as that containing the GLUT4 promoter) and toregulate insulin adipocytes and production. The DMEF1 response elementis present in the GLUT4 promoter and binds to MEF2 pre-adipocytestranscription factor and another transcription factor that is requiredfor insulin regulation of Glut4 expression in skeletal muscle. GLUT4 isthe primary insulin-responsive glucose transporter in fat and muscletissue. Exemplary assays that may be used or routinely modified to testfor DMEF1 response element activity (in adipocytes and pre-adipocytes)by polypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed inThai, M. V., etal., J Biol Chem, 273(23): 14285-92 (1998); Mora, S., et al., J BiolChem, 275(21): 16323-8 (2000); Liu, M. L., et al., J Biol Chem, 269(45):28514-21 (1994); “Identification of a 30-base pair regulatory elementand novel DNA binding protein that regulates the human GLUT4 promoter intransgenic mice”, J Biol Chem. 2000 Aug 4; 275(31): 23666-73; Berger, etal., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol.216: 362-368 (1992), the contents of each of which is hereinincorporated by reference in its entirety. Adipocytes and pre-adipocytesthat may be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include the mouse 3T3-L1 cellline which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1cells are a continuous substrain of 3T3 fibroblasts developed throughclonal isolation. These cells undergo a pre-adipocyte to adipose-likeconversion under appropriate differentiation culture conditions. 172HUKDF20 376 Activation of Assays for the activation of transcriptionthrough the AP1 response element are known in the art and transcriptionmay be used or routinely modified to assess the ability of polypeptidesof the invention (including through AP1 antibodies and agonists orantagonists of the invention) to modulate growth and other cellfunctions. response element Exemplary assays for transcription throughthe AP1 response element that may be used or routinely in immune cellsmodified to test AP1-response element activity of polypeptides of theinvention (including (such as T-cells). antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1988); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Rellahan et al., J Biol Chem 272(49): 30806-30811 (1997); Chang et al.,Mol Cell Biol 18(9): 4986-4993 (1998); and Fraser et al., Eur J Immunol29(3): 838-844 (1999), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary mouse T cells that may be used according to theseassays include the CTLL cell line, which is an IL-2 dependentsuspension-culture cell line with cytotoxic activity. 172 HUKDF20 376Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate the serum responseelement response factors and modulate the expression of genes involvedin growth. Exemplary assays for in immune cells transcription throughthe SRE that may be used or routinely modified to test SRE activity ofthe (such as T-cells). polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66 :1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); and Black et al., Virus Genes 12(2):105-117 (1997), the content of each of which are herein incorporated byreference in its entirety. T cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseT cells that may be used according to these assays include the CTLL cellline, which is an IL-2 dependent suspension culture of T cells withcytotoxic activity. 172 HUKDF20 376 Activation of Assays for theactivation of transcription through the NFKB response element arewell-known in transcription the art and may be used or routinelymodified to assess the ability of polypeptides of the invention throughNFKB (including antibodies and agonists or antagonists of the invention)to regulate NFKB transcription response element factors and modulateexpression of immunomodulatory genes. Exemplary assays for transcriptionin immune cells through the NFKB response element that may be used orrountinely modified to test NFKB- (such as EOL1 response elementactivity of polypeptides of the invention (including antibodies andagonists or cells). antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3):455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); andFraser et al., 29(3): 838-844 (1999), the contents of each of which areherein incorporated by reference in its entirety. For example, areporter assay (which measures increases in transcription inducible froma NFkB responsive element in EOL-1 cells) may link the NFKB element to arepeorter gene and binds to the NFKB transcription factor, which isupregulated by cytokines and other factors. Exemplary immune cells thatmay be used according to these assays include eosinophils such as thehuman EOL-1 cell line of eosinophils. Eosinophils are a type of immunecell important in the allergic responses; they are recruited to tissuesand mediate the inflammtory response of late stage allergic reaction.Eol-1 is a human eosinophil cell line. 172 HUKDF20 376 Activation ofThis reporter assay measures activation of the NFkB signaling pathway inKu812 human basophil transcription cell line. Assays for the activationof transcription through the NFKB response element are well- throughNFKB known in the art and may be used or routinely modified to assessthe ability of polypeptides of the response element invention (includingantibodies and agonists or antagonists of the invention) to regulateNFKB in immune cells transcription factors and modulate expression ofimmunomodulatory genes. Exemplary assays for (such as transcriptionthrough the NFKB response element that may be used or rountinelymodified to test basophils). NFKB-response element activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), thecontents of each of which are herein incorporated by reference in itsentirety. Basophils that may be used according to these assays arepublicly available (e.g., through the ATCC). Exemplary human basophilcell lines that may be used according to these assays include Ku812,originally established from a patient with chronic myelogenous leukemia.It is an immature prebasophilic cell line that can be induced todifferentiate into mature basophils. 172 HUKDF20 376 Production ofAssays for measuring expression of ICAM-1 are well-known in the art andmay be used or routinely ICAM-1 modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) to regulate ICAM-1 expression. Exemplaryassays that may be used or routinely modified to measure ICAM-1expression include assays disclosed in: Takacs P, et al, FASEB J, 15(2):279-281 (2001); and, Miyamoto K, et al., Am J Pathol, 156(5): 1733-1739(2000), the contents of each of which is herein incorporated byreference in its entirety. Cells that may be used according to theseassays are publicly available (e.g., through the ATCC) and/or may beroutinely generated. Exemplary cells that may be used according to theseassays include microvascular endothelial cells (MVEC). 172 HUKDF20 376Activation of Assays for the activation of transcription through theSerum Response Element (SRE) are well- transcription known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the through serum invention (including antibodies andagonists or antagonists of the invention) to regulate serum responseelement response factors and modulate the expression of genes involvedin growth and upregulate the in immune cells function of growth-relatedgenes in many cell types. Exemplary assays for transcription through the(such as natural SRE that may be used or routinely modified to test SREactivity of the polypeptides of the invention killer cells). (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Benson et al., J Immunol 153(9): 3862-3873(1994); and Black et al., Virus Genes 12(2): 105-117 (1997), the contentof each of which are herein incorporated by reference in its entirety. Tcells that may be used according to these assays are publicly available(e.g., through the ATCC). Exemplary T cells that may be used accordingto these assays include the NK-YT cell line, which is a human naturalkiller cell line with cytolytic and cytotoxic activity. 173 HUSCJ14 377Regulation of Assays for the regulation of transcription through the FASpromoter element are well-known in the transcription art and may be usedor routinely modified to assess the ability of polypeptides of theinvention through the FAS (including antibodies and agonists orantagonists of the invention) to activate the FAS promoter promoterelement element in a reporter construct and to regulate transcription ofFAS, a key enzyme for lipogenesis. in hepatocytes FAS promoter isregulated by many transcription factors including SREBP. Insulinincreases FAS gene transcription in livers of diabetic mice. Thisstimulation of transcription is also somewhat glucose dependent.Exemplary assays that may be used or routinely modified to test for FASpromoter element activity (in hepatocytes) by polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in Xiong, S., et al., Proc Natl AcadSci U.S.A., 97(8): 3948-53 (2000); Roder, K., et al., Eur J Biochem,260(3): 743-51 (1999); Oskouian B, et al., Biochem J, 317 (Pt 1): 257-65(1996); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al.,Methods in Enzymol. 216: 362-368 (1992), the contents of each of whichis herein incorporated by reference in its entirety. Hepatocytes thatmay be used according to these assays, such as H4IIE cells, are publiclyavailable (e.g., through the ATCC) and/or may be routinely generated.Exemplary hepatocytes that may be used according to these assays includerat liver hepatoma cell line(s) inducible with glucocorticoids, insulin,or cAMP derivatives. 173 HUSCJ14 377 Upregulation of CD71 FMAT. CD71 isthe transferrin receptor. Transferrin is a major iron carrying proteinthat is CD71 and essential for cell proliferation. CD71 is expressedpredominantly on cells that are actively activation of proliferating.Assays for immunomodulatory proteins expressed on activated T cells, Bcells, and T cells most proliferating cells are well known in the artand may be used or routinely modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) to modulate the activation of T cells,and/or mediate humoral or cell-mediated immunity. Exemplary assays thattest for immunomodulatory proteins evaluate the upregulation of cellsurface markers, such as CD71, and the activation of T cells. Suchassays that may be used or routinely modified to test immunomodulatoryactivity of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include, for example, theassays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204(1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6:138-160 (2000); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993),the contents of each of which are herein incorporated by reference inits entirety. Human T cells that may be used according to these assaysmay be isolated using techniques disclosed herein or otherwise known inthe art. Human T cells are primary human lymphocytes that mature in thethymus and express a T Cell receptor and CD3, CD4, or CD8. These cellsmediate humoral or cell-mediated immunity and may be preactivated toenhance responsiveness to immunomodulatory factors. 174 HUVDJ48 378Regulation of Assays for the regulation of viability and proliferationof cells in vitro are well-known in the art and viability and may beused or routinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 174 HUVDJ48 378 Activation of Kinase assay.JNK kinase assays for signal transduction that regulate cellproliferation, activation, JNK Signaling or apoptosis are well known inthe art and may be used or routinely modified to assess the abilityPathway in of polypeptides of the invention (including antibodies andagonists or antagonists of the invention) immune cells to promote orinhibit cell proliferation, activation, and apoptosis. Exemplary assaysfor JNK kinase (such as activity that may be used or routinely modifiedto test JNK kinase-induced activity of polypeptides eosinophils). of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Gupta et al., Exp Cell Res 247(2): 495-504(1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin,Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol71(3-4): 479-500 (1999); the contents of each of which are hereinincorporated by reference in its entirety. Exemplary cells that may beused according to these assays include eosinophils. Eosinophils areimportant in the late stage of allergic reactions; they are recruited totissues and mediate the inflammatory response of late stage allergicreaction. Moreover, exemplary assays that may be used or routinelymodified to assess the ability of polypeptides of the invention(including antibodies and agonists or antagonists of the invention) tomodulate signal transduction, cell proliferation, activation, orapoptosis in eosinophils include assays disclosed and/or cited in: ZhangJP, et al., “Role of caspases in dexamethasone-induced apoptosis andactivation of c-Jun NH2-terminal kinase and p38 mitogen-activatedprotein kinase in human eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7(2000); Hebestreit H, et al., “Disruption of fas receptor signaling bynitric oxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); JAllergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, etal., “In vivo resistance to corticosteroids in bronchial asthma isassociated with enhanced phosyphorylation of JUN N-terminal kinase andfailure of prednisolone to inhibit JUN N-terminal kinasephosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74(1999); the contents of each of which are herein incorporated byreference in its entirety. 175 HBDAB91 379 Stimulation of Assays formeasuring secretion of insulin are well-known in the art and may be usedor routinely insulin secretion modified to assess the ability ofpolypeptides of the invention (including antibodies and agonists or frompancreatic antagonists of the invention) to stimulate insulin secretion.For example, insulin secretion is beta cells. measured by FMAT usinganti-rat insulin antibodies. Insulin secretion from pancreatic betacells is upregulated by glucose and also by certain proteins/peptides,and disregulation is a key component in diabetes. Exemplary assays thatmay be used or routinely modified to test for stimulation of insulinsecretion (from pancreatic cells) by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Ahren, B., et al., Am J Physiol, 277(4 Pt2): R959-66 (1999); Li, M., et al., Endocrinology, 138(9): 3735-40(1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9 (1995); and,Miraglia S et. al., Journal of Biomolecular Screening, 4: 193-204(1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 176 HELGG84 380 Activation of Kinase assay.Kinase assays, for example an Elk-1 kinase assay, for ERK signaltransduction that Adipocyte ERK regulate cell proliferation ordifferentiation are well known in the art and may be used or routinelySignaling modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or Pathway antagonists ofthe invention) to promote or inhibit cell proliferation, activation, anddifferentiation. Exemplary assays for ERK kinase activity that may beused or routinely modified to test ERK kinase-induced activity ofpolypeptides of the invention (including antibodies and agonists orantagonists of the invention) include the assays disclosed in Forrer etal., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, ExpClin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, BiochemSoc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40(2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); thecontents of each of which are herein incorporated by reference in itsentirety. Mouse adipocyte cells that may be used according to theseassays are publicly available (e.g., through the ATCC). Exemplary mouseadipocyte cells that may be used according to these assays include3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that isa continuous substrain of 3T3 fibroblast cells developed through clonalisolation and undergo a pre-adipocyte to adipose-like conversion underappropriate differentiation conditions known in the art. 177 HILCA24 381Regulation of Assays for the regulation of viability and proliferationof cells in vitro are well-known in the art and viability and may beused or routinely modified to assess the ability of polypeptides of theinvention (including proliferation of antibodies and agonists orantagonists of the invention) to regulate viability and proliferation ofpancreatic beta pancreatic beta cells. For example, the Cell Titer-Gloluminescent cell viability assay measures the cells. number of viablecells in culture based on quantitation of the ATP present which signalsthe presence of metabolically active cells. Exemplary assays that may beused or routinely modified to test regulation of viability andproliferation of pancreatic beta cells by polypeptides of the invention(including antibodies and agonists or antagonists of the invention)include assays disclosed in: Friedrichsen BN, et al., Mol Endocrinol,15(1): 136-48 (2001); Huotari MA, et al., Endocrinology, 139(4): 1494-9(1998); Hugl SR, et al., J Biol Chem 1998 Jul 10; 273(28): 17771-9(1998), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167. 177 HILCA24 381 Activation of Assays forthe activation of transcription through the Signal Transducers andActivators of transcription Transcription (STAT6) response element arewell-known in the art and may be used or routinely through STAT6modified to assess the ability of polypeptides of the invention(including antibodies and agonists or response element antagonists ofthe invention) to regulate STAT6 transcription factors and modulate theexpression in immune cells of multiple genes. Exemplary assays fortranscription through the STAT6 response element that (such as T-cells).may be used or routinely modified to test STAT6 response elementactivity of the polypeptides of the invention (including antibodies andagonists or antagonists of the invention) include assays disclosed inBerger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85:6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffattet al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur JImmunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38):29331-29337 (2000), the contents of each of which are hereinincorporated by reference in its entirety. T cells that may be usedaccording to these assays are publicly available (e.g., through theATCC). Exemplary T cells that may be used according to these assaysinclude the HT2 cell line, which is an IL-2 dependent suspension cultureof T cells that also respond to IL-4. 177 HILCA24 381 Activation ofAssays for the activation of transcription through the SignalTransducers and Activators of transcription Transcription (STAT6)response element are well-known in the art and may be used or routinelythrough STAT6 modified to assess the ability of polypeptides of theinvention (including antibodies and agonists or response elementantagonists of the invention) to regulate STAT6 transcription factorsand modulate the expression in immune cells of multiple genes. Exemplaryassays for transcription through the STAT6 response element that (suchas T-cells). may be used or routinely modified to test STAT6 responseelement activity of the polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) include assaysdisclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm,Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl AcadSci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538(1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curielet al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J BiolChem 275(38): 29331-29337 (2000), the contents of each of which areherein incorporated by reference in its entirety. T cells that may beused according to these assays are publicly available (e.g., through theATCC). Exemplary T cells that may be used according to these assaysinclude the SUPT cell line, which is a suspension culture of IL-2 andIL-4 responsive T cells. 178 HYABC84 382 Stimulation of Assays formeasuring calcium flux are well-known in the art and may be used orroutinely Calcium Flux in modified to assess the ability of polypeptidesof the invention (including antibodies and agonists or pancreatic betaantagonists of the invention) to mobilize calcium. For example, the FLPRassay may be used to cells. measure influx of calcium. Cells normallyhave very low concentrations of cytosolic calcium compared to muchhigher extracellular calcium. Extracellular factors can cause an influxof calcium, leading to activation of calcium responsive signalingpathways and alterations in cell functions. Exemplary assays that may beused or routinely modified to measure calcium flux by polypeptides ofthe invention (including antibodies and agonists or antagonists of theinvention) include assays disclosed in: Satin LS, et al., Endocrinology,136(10): 4589-601 (1995); Mogami H, et al., Endocrinology, 136(7):2960-6 (1995); Richardson SB, et al., Biochem J, 288 (Pt 3): 847-51(1992); and, Meats, JE, et al., Cell Calcium 1989 Nov-Dec; 10(8): 535-41(1989), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include HITT15 Cells. HITT15 are an adherentepithelial cell line established from Syrian hamster islet cellstransformed with SV40. These cells express glucagon, somatostatin, andglucocorticoid receptors. The cells secrete insulin, which is stimulatedby glucose and glucagon and suppressed by somatostatin orglucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J.219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343,1981. 179 HMCAZ04 383 Activation of Kinase assay. Kinase assays, forexample an Elk-1 kinase assay, for ERK signal transduction thatAdipocyte ERK regulate cell proliferation or differentiation are wellknown in the art and may be used or routinely Signaling modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or Pathway antagonists of the invention) topromote or inhibit cell proliferation, activation, and differentiation.Exemplary assays for ERK kinase activity that may be used or routinelymodified to test ERK kinase-induced activity of polypeptides of theinvention (including antibodies and agonists or antagonists of theinvention) include the assays disclosed in Forrer et al., Biol Chem379(8-9): 1101-1110 (1998); Le Marchand-Brustel Y, Exp Clin EndocrinolDiabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48(1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH,Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each ofwhich are herein incorporated by reference in its entirety. Mouseadipocyte cells that may be used according to these assays are publiclyavailable (e.g., through the ATCC). Exemplary mouse adipocyte cells thatmay be used according to these assays include 3T3-L1 cells. 3T3-L1 is anadherent mouse preadipocyte cell line that is a continuous substrain of3T3 fibroblast cells developed through clonal isolation and undergo apre-adipocyte to adipose-like conversion under appropriatedifferentiation conditions known in the art. 180 HE8FD92 384 Stimulationof Assays for measuring secretion of insulin are well-known in the artand may be used or routinely insulin secretion modified to assess theability of polypeptides of the invention (including antibodies andagonists or from pancreatic antagonists of the invention) to stimulateinsulin secretion. For example, insulin secretion is beta cells.measured by FMAT using anti-rat insulin antibodies. Insulin secretionfrom pancreatic beta cells is upregulated by glucose and also by certainproteins/peptides, and disregulation is a key component in diabetes.Exemplary assays that may be used or routinely modified to test forstimulation of insulin secretion (from pancreatic cells) by polypeptidesof the invention (including antibodies and agonists or antagonists ofthe invention) include assays disclosed in: Ahren, B., et al., Am JPhysiol, 277(4 Pt 2): R959-66 (1999); Li, M., et al., Endocrinology,138(9): 3735-40 (1997); Kim, K. H., et al., FEBS Lett, 377(2): 237-9(1995); and, Miraglia S et. al., Journal of Biomolecular Screening, 4:193-204 (1999), the contents of each of which is herein incorporated byreference in its entirety. Pancreatic cells that may be used accordingto these assays are publicly available (e.g., through the ATCC) and/ormay be routinely generated. Exemplary pancreatic cells that may be usedaccording to these assays include rat INS-1 cells. INS-1 cells are asemi-adherent cell line established from cells isolated from an X-rayinduced rat transplantable insulinoma. These cells retaincharacteristics typical of native pancreatic beta cells includingglucose inducible insulin secretion. References: Asfari et al.Endocrinology 1992 130: 167.

Table 2 further characterizes certain encoded polypeptides of theinvention, by providing the results of comparisons to protein andprotein family databases. The first column provides a unique cloneidentifier, “Clone ID NO:”, corresponding to a cDNA clone disclosed inTable 1A and/or Table 1B. The second column provides the unique contigidentifier, “Contig ID:” which allows correlation with the informationin Table 1B. The third column provides the sequence identifier, “SEQ IDNO:”, for the contig polynucleotide sequences. The fourth columnprovides the analysis method by which the homology/identity disclosed inthe Table was determined. The fifth column provides a description of thePFAM/NR hit identified by each analysis. Column six provides theaccession number of the PFAM/NR hit disclosed in the fifth column.Column seven, score/percent identity, provides a quality score or thepercent identity, of the hit disclosed in column five. Comparisons weremade between polypeptides encoded by polynucleotides of the inventionand a non-redundant protein database (herein referred to as “NR”), or adatabase of protein families (herein referred to as “PFAM”), asdescribed below.

The NR database, which comprises the NBRF PIR database, the NCBI GenPeptdatabase, and the SIB SwissProt and TrEMBL databases, was madenon-redundant using the computer program nrdb2 (Warren Gish, WashingtonUniversity in Saint Louis). Each of the polynucleotides shown in Table1B, column 3 (e.g., SEQ ID NO:X or the ‘Query’ sequence) was used tosearch against the NR database. The computer program BLASTX was used tocompare a 6-frame translation of the Query sequence to the NR database(for information about the BLASTX algorithm please see Altshul et al.,J. Mol. Biol. 215:403410 (1990), and Gish and States, Nat. Genet.3:266-272 (1993). A description of the sequence that is most similar tothe Query sequence (the highest scoring ‘Subject’) is shown in columnfive of Table 2 and the database accession number for that sequence isprovided in column six. The highest scoring ‘Subject’ is reported inTable 2 if (a) the estimated probability that the match occurred bychance alone is less than 1.0e-07, and (b) the match was not to a knownrepetitive element. BLASTX returns alignments of short polypeptidesegments of the Query and Subject sequences which share a high degree ofsimilarity; these segments are known as High-Scoring Segment Pairs orHSPs. Table 2 reports the degree of similarity between the Query and theSubject for each HSP as a percent identity in Column 7. The percentidentity is determined by dividing the number of exact matches betweenthe two aligned sequences in the HSP, dividing by the number of Queryamino acids in the HSP and multiplying by 100. The polynucleotides ofSEQ ID NO:X which encode the polypeptide sequence that generates an HSPare delineated by columns 8 and 9 of Table 2.

The PFAM database, PFAM version 2.1, (Sonnhammer, Nucl. Acids Res.,26:320-322, 1998))consists of a series of multiple sequence alignments;one alignment for each protein family. Each multiple sequence alignmentis converted into a probability model called a Hidden Markov Model, orHMM, that represents the position-specific variation among the sequencesthat make up the multiple sequence alignment (see, e.g., Durbin, et al.,Biological sequence analysis: probabilistic models of proteins andnucleic acids, Cambridge University Press, 1998 for the theory of HMMs).The program HMMER version 1.8 (Sean Eddy, Washington University in SaintLouis) was used to compare the predicted protein sequence for each Querysequence (SEQ ID NO:Y in Table 1B) to each of the HMMs derived from PFAMversion 2.1. A HMM derived from PFAM version 2.1 was said to be asignificant match to a polypeptide of the invention if the scorereturned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 scoreobtained with the most distantly related known member of that proteinfamily. The description of the PFAM family which shares a significantmatch with a polypeptide of the invention is listed in column 5 of Table2, and the database accession number of the PFAM hit is provided incolumn 6. Column 7 provides the score returned by HMMER version 1.8 forthe alignment. Columns 8 and 9 delineate the polynucleotides of SEQ IDNO:X which encode the polypeptide sequence which show a significantmatch to a PFAM protein family.

As mentioned, columns 8 and 9 in Table 2, “NT From” and “NT To”,delineate the polynucleotides of “SEQ ID NO:X” that encode a polypeptidehaving a significant match to the PFAM/NR database as disclosed in thefifth column. In one embodiment, the invention provides a proteincomprising, or alternatively consisting of, a polypeptide encoded by thepolynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2.Also provided are polynucleotides encoding such proteins, and thecomplementary strand thereto.

The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y aresufficiently accurate and otherwise suitable for a variety of uses wellknown in the art and described further below. For instance, thenucleotide sequences of SEQ ID NO:X are useful for designing nucleicacid hybridization probes that will detect nucleic acid sequencescontained in SEQ ID NO:X or the cDNA contained in ATCC Deposit No:Z.These probes will also hybridize to nucleic acid molecules in biologicalsamples, thereby enabling immediate applications in chromosome mapping,linkage analysis, tissue identification and/or typing, and a variety offorensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used to generateantibodies which bind specifically to these polypeptides, or fragmentsthereof, and/or to the polypeptides encoded by the cDNA clonesidentified in, for example, Table 1A and/or 1B.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X, and a predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing cDNA ATCC DepositNo:Z (e.g., as set forth in columns 2 and 3 of Table 1A and/or as setforth, for example, in Table 1B, 6, and 7). The nucleotide sequence ofeach deposited clone can readily be determined by sequencing thedeposited clone in accordance with known methods. Further, techniquesknown in the art can be used to verify the nucleotide sequences of SEQID NO:X. The predicted amino acid sequence can then be verified fromsuch deposits. Moreover, the amino acid sequence of the protein encodedby a particular clone can also be directly determined by peptidesequencing or by expressing the protein in a suitable host cellcontaining the deposited human cDNA, collecting the protein, anddetermining its sequence. TABLE 2 SEQ ID PFam/NR Score/ cDNA CloneContig NO: Analysis Accession Percent NT NT ID: ID X Method PFam/NRDescription Number Identity From To H6EDM64 841331 11 WUblastx.64(Q9UID3) ANG2. Q9UID3  90% 928 2451  36% 203 310  36% 931 1038  95% 191871 HACBJ56 847112 14 WUblastx.64 (Q9D2Q2) 2310079F23RIK PROTEIN. Q9D2Q2 65% 98 286 HADMB15 847116 15 WUblastx.64 (Q9BVH1) SIMILAR TO DLXIN-1.Q9BVH1 100% 8 109 HAJBV67 866415 18 WUblastx.64 (Q9HD45) TRANSMEMBRANE 9T9S3_HUMAN 100% 13 126 SUPERFAMILY PROTEIN MEMBER 3 PRECU  93% 116 1681HAOAG15 852204 20 HMMER PFAM: von Willebrand factor type A domainPF00092 180.1 506 1057 2.1.1 WUblastx.64 (O75578) INTEGRIN ALPHA-10PRECURSOR. ITAG_HUMAN  90% 8 3463 HATCI03 580805 22 WUblastx.64 (Q9H743)CDNA: FLJ21394 FIS, CLONE Q9H743  71% 906 688 COL03536. HBAGD86 83879923 WUblastx.64 (Q14287) HYPOTHETICAL PROTEIN Q14287  37% 801 559(FRAGMENT). HBHAA05 603174 24 WUblastx.64 (Q9H387) PR02550. Q9H387  71%676 386 HBHAA81 846465 25 WUblastx.64 (Q9D1G3) 1110011D13RJK PROTEIN.Q9D1G3  89% 1329 1502  79% 28 1329 HBJAC40 841235 27 WUblastx.64(Q9P112) CHROMOSOME 16 OPEN READING Q9P112 100% 8 73 FRAME 5.  36% 5 70 57% 11 52  53% 85 180 100% 192 632 HBJCR46 815649 28 HMMER PFAM: WDdomain, G-beta repeat PF00400 36.6 790 867 2.1.1 WUblastx.64 (Q9DC22)1200006M05RIK PROTEIN. Q9DC22  96% 207 611  73% 568 2763 HBJEL16 84703030 WUblastx.64 (095297) PROTEIN ZERO RELATED 095297  98% 285 491PROTEIN. HBJIG20 866159 31 HMMER PFAM: Cytochrome c oxidase subunit IIIPF00510 162.6 321 551 2.1.1 WUblastx.64 (BAA77671) Cytochrome c oxidasesubunit 3 BAA77671  81% 9 617 (Fragment HBJKD16 853358 32 WUblastx.64(Q9NXS4) CDNA FLJ20080 FIS, CLONE Q9NXS4  91% 8 1528 COLO3184. HBMTY48637521 33 WUblastx.64 (Q9H5N9) CDNA: FLJ23235 FIS, CLONE Q9H5N9  94% 54941 CAS04980. HBMUH74 866160 34 WUblastx.64 (Q9NVW8) CDNA FLJ10462 FIS,CLONE Q9NVW8 100% 11 427 NT2RP1001494, WEAKLY SIMILAR TO MAL HBQAB79810542 35 WUblastx.64 (Q9UQ32) AD 3 (FRAGMENT). Q9UQ32  82% 323 204HBXCX15 637542 36 WUblastx.64 (Q9GMX5) HYPOTHETICAL 12.9 KDA Q9GMX5  41%726 827 PROTEIN.  52% 578 730 HCDCY76 837972 37 WUblastx.64 frizzledprotein 4 - human pir|JC7127|JC7127 100% 1039 527  30% 994 785  79% 56737 HCEDR26 771144 38 WUblastx.64 (Q9H919) CDNA FLJ13078 FIS, CLONEQ9H919  66% 1157 1095 NT2RP3002002.  66% 1345 1184 HCEEU18 688041 39WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083  49% 186 10  56%1223 933 HCFLN88 610000 41 WUblastx.64 (Q9BQE9) SIMILAR TO B-CELL Q9BQE9 87% 278 475 CLL/LYMPHOMA 7B (UNKNOWN) (PROTEIN FOR MGC HCHAB84 83432642 WUblastx.64 (Q9BRV3) STROMAL CELL PROTEIN. Q9BRV3  89% 82 744 HCNSD29862314 45 WUblastx.64 (O75400) HUNTINGTIN-INTERACTING Q75400  82% 6281605 PROTEIN HYPA/FBP11 (FRAGMENT).  78% 337 489 HDPDJ58 587265 51WUblastx.64 hypothetical protein DKFZp434D2328.1 - humanpir|T42691|T42691 100% 307 609 (fragment)  87% 621 785 HDPFU43 790189 52WUblastx.64 (AAH01057) Tyrosylprotein sulfotransferase 2. AAH01057 100%360 1349 58% 220 348 HDPIU94 813352 54 WUblastx.64 (Q9BVF7) SIMILAR TOHYPOTHETICAL Q9BVF7  99% 63 1703 PROTEIN FLJ10422. HDPIY31 886159 55WUblastx.64 hypothetical protein DKFZp434N1429.1 - humanpir|T46448|T46448  72% 1714 1899 (fragment) HDPOC24 777493 56WUblastx.64 (Q9H8K1) CDNA FLJ13518 FIS, CLONE Q9H8K1 100% 62 208PLACE1005799. HDPPQ30 684292 58 WUblastx.64 (Q9H387) PR02550. Q9H387 51% 807 727  79% 1042 815 HDQHM36 852328 59 WUblastx.64 (Q9N083)UNNAMED PORTEIN PRODUCT. Q9N083  69% 1129 1257  50% 965 1153 HE2HC60753265 62 WUblastx.64 (Q9NVC4) CDNA FLJ10814 FIS, CLONE Q9NVC4  88% 1251300 NT2RP4000984. HE6FU11 827236 64 HMMER PFAM: von Willebrand factortype A domain PF00092 184.7 244 771 2.1.1 WUblastx.64 (095460)MATRILIN-4 PRECURSOR. MTN4_HUMAN  77% 145 789  45% 782 907  41% 791 925 50% 794 907  38% 863 1498  33% 190 741  98% 782 1642 HE8TY46 899528 66WUblastx.64 (BAB55144) CDNA FLJ14576 fis, clone BAB55144  95% 318 938NT2RM4001092, w HE9EA10 827796 67 WUblastx.64 laminin alpha-1 chainprecursor - human pir|S14458|S14458  99% 761 1891  27% 878 1840  25%1142 1876 HEBFR46 847064 69 WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS,CLONE Q9NX85  80% 1111 1022 KAIA0536  84% 1265 1110 HEBGE07 798096 70WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS, CLONE Q9NX85  79% 1851 1720KAIA0536. HETCI16 844543 72 WUblastx.64 (Q9P0V3) BOG25. Q90V3  99% 3 356HFCFE20 701985 74 WUblastx.64 (Q9CSE5) EUKARYOTIC TRANSLATION Q9CSE5 89% 438 581 INITIATION FACTOR 3 (FRAGMENT).  54% 1083 1187 HFPDS07821646 77 WUblastx.64 (O94925) GLUTAMINASE, KIDNEY ISOFORM, GLSK_HUMAN 78% 343 513 MITOCHONDRIAL PRECURS  74% 2 436 HFVHW43 570948 78WUblastx.64 (Q9BGX4) HYPOTHETICAL 13.8 KDA Q9BGX4  69% 1209 1093PROTEIN. HGBHP91 693011 81 WUblastx.64 hypothetical protein (L1H3′region) - human pir|B34087|B34087  52% 541 491  44% 537 34 HHEAK45765278 82 WUblastx.64 (Q9NPB0) DJ202I21.1 (NOVEL PROTEIN) Q9NPB0  68%1949 1458 (CDNA FLJ11101 FIS, CLONE PLACE10 HHEOW19 886174 83WUblastx.64 (018973) RAB5 GDP/GTP EXCHANGE 018973  77% 417 623 FACTOR,RABEX5.  91% 611 715  56% 166 378  92% 129 167 HHFFS40 824059 84WUblastx.64 (Q9H4A6) GOLGI PROTEIN. Q9H4A6 100% 3 251 HHSBI65 801910 88WUblastx.64 (Q9H5W9) CDNA:FLJ22888 FIS, CLONE Q9H5W9 100% 270 407KAT03934.  94% 479 1300 HISAT67 843549 90 WUblastx.64 (Q9UH94) PROLACTINREGULATORY Q9UH94  88% 219 797 ELEMENT-BINDING PROTEIN (PROLACTIN  91%788 1447 REGU HJBCU75 638329 91 WUblastx.64 (O45030) STRABISMUS. 045030 44% 199 426  52% 464 964 HJMAA03 824062 92 WUblastx.64 (Q9N032) UNNAMEDPROTEIN PRODUCT. Q9N032  71% 415 528 HKABU43 838573 93 WUblastx.64(AAH03633) Translocase of outer mitochondrial AAH03633 100% 33 62 membr 92% 26 1597 HKACI79 853361 94 WUblastx.64 (Q9BGV8) HYPOTHETICAL 10.0KDA Q9BGV8  72% 886 1104 PROTEIN. HLDQU79 740755 97 WUblastx.64 (O75477)KE04P. 075477 100% 105 1142 HLDQU79 837599 191 blastx.2 KE04P.sp|O75477|O75477  99% 81 1118 HLHAP05 638476 98 WUblastx.64 (Q9HA67)CDNA FLJ12155 FIS, CLONE Q9HA67  55% 1553 1500 MAMMA1000472.  72% 16501585  77% 1807 1646 HLICE88 840321 100 WUblastx.64 fibrinogen gamma-Achain precursor [validated] - pir|A90470|FGHUG 89% 3 584 human HLMGP50647603 101 WUblastx.64 (Q9GMI7) HYPOTHETICAL 9.0 KDA Q9GMI7  61% 765 709PROTEIN. 72% 935 807 HLQCX36 584786 103 WUblastx.64 (Q9UI59) PRO0478PROTEIN. Q9UI59  87% 1100 1216 HLWDB73 838453 105 WUblastx.64 (Q9H7D7)CDNA: FLJ21016 FIS, CLONE Q9H7D7 100% 660 872 CAE05735. 98% 1 657HLYGB19 838083 107 WUblastx.64 (Q9H0Q1) HYPOTHETICAL 12.3 KDA Q9H0Q1 97% 204 518 PROTEIN. HLYGY91 658703 108 WUblastx.64 (Q9H8N0) CDNAFLJ13386 FIS, CLONE Q9H8N0  94% 221 391 PLACE1001104, WEAKLY SIMILAR TOMYO HMDAB29 584789 109 WUblastx.64 (Q9NX17) CDNA FLJ20489 FIS, CLONEQ9NX17  72% 1186 890 KAT08285. HMEDI90 840077 111 WUblastx.64 (Q9HBA3)RAB3 INTERACTING PROTEIN Q9HBA3 100% 81 794 VARIANT 4 (FRAGMENT).HMICP65 847403 113 WUblastx.64 (Q9HAU9) GUANINE NUCLEOTIDE BINDINGQ9HAU9  99% 8 892 PROTEIN BETA SUBUNIT 5L.  22% 269 943 HMSHC86 840402114 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083  70% 1724 1674 67% 1674 1420 HMUAN45 833072 115 WUblastx.64 (BAB55441) CDNA FLJ14993fis, clone BAB55441  70% 684 1238 Y79AA1001874, w  65% 239 955 100% 12471516 HMVBC31 825598 116 WUblastx.64 (O60725) PROTEIN-S ISOPRENYLCYSTEINEICMT_HUMAN  80% 747 938 O-METHYLTRANSFERASE (E  87% 121 789 HMWBL03822861 117 WUblastx.64 (Q9BWT1) C-MYC TARGET JP1. Q9BWT1  85% 137 1240HNGAK51 603910 119 WUblastx.64 (O60448) NEURONAL THREAD PROTEIN O60448 61% 563 601 AD7C-NTP.  67% 733 915  65% 702 878  74% 714 914 HNGGP65597449 121 WUblastx.64 (Q9GMU5) HYPOTHETICAL 14.1 KDA Q9GMU5  31% 69 302PROTEIN.  47% 398 541 HNHFE71 834487 126 WUblastx.64 hypotheticalprotein DKFZp761L0812.1 - human pir|T47135|T47135  67% 822 583 fragment)HOACG07 792928 128 WUblastx.64 (Q9GZN8) DJ1009E24.3 (A NOVEL PROTEIN)Q9GZN8  99% 183 704 (CDNA FLJ14158 FIS, CLONE NT2R HOEBK60 789396 130WUblastx.64 (Q9H916) CDNA FL113081 FIS, CLONE Q9H916  98% 132 1916NT2RP3002033. 100% 14 109  88% 106 159 HOFAA78 836646 131 WUblastx.64(Q9NXS2) CDNA FLJ20084 FIS, CLONE Q9NXS2  90% 529 792 COL03526.  50% 980  88% 29 529 HOFNB74 762821 132 WUblastx.64 (Q99JH1) HYPOTHETICAL 17.7KDA Q99JH1  72% 44 187 PROTEIN.  97% 199 471 HOSDO75 862049 134WUblastx.64 (Q9D099) 1110057LI8RIK PROTEIN. Q9D099  89% 11 202  88% 259630 HOUDR07 745404 135 WUblastx.64 (Q9HBV4) ANGIOPOIETIN-LIKE PROTEIN9HBV4  87% 170 1384 PP1158. HPFCI36 855966 137 WUblastx.64 (Q9NX47) CDNAFLJ20445 FIS, CLONE Q9NX47 100% 9 320 KAT05170. HPRBH85 695752 141WUblastx.64 (BAB55300) CDNA FLJ14784 fis, clone BAB55300  62% 2 616NT2RP4000713.  86% 534 1085 HPRCA64 824074 142 WUblastx.64 (P55161)NCK-ASSOCIATED PROTEIN 1 (NAP1) NCP1_RAT 100% 1021 1926 (P12SNAP1)(MEMBR  85% 387 1019  93% 11 481 HPRCD35 853551 143 WUblastx.64hypothetical protein DKFZp762L1710.1 - human pir|T50629|T50629 100% 320613 (fragment)  57% 2 499 HPTRM02 812879 144 WUblastx.64 (Q9UJU6) SRCHOMOLOGY 3 DOMAIN- Q9UJU6  92% 332 940 CONTAINING PROTEIN HIP-55(DREBRIN F).  97% 2 106  96% 98 190 HRAAD30 866187 145 WUblastx.64(Q9H6V0) CDNA: FLJ21839 FIS, CLONE Q9H6V0  89% 23 1393 HEP01794. HRADF49866481 146 WUblastx.64 (Q9H6L1) CDNA: FLJ22169 FIS, CLONE Q9H6L1  90% 13825 HRC00632.  84% 813 1379  75% 1291 1593  34% 1590 1685 HRDDQ39 840405147 WUblastx.64 (Q9NX85) CDNA FLJ20378 FIS, CLONE Q9NX85  53% 582 436KAIA0536.  65% 775 578 HRDEX93 816046 148 WUblastx.64 (Q9UBV8) PEFLIN.Q9UBV8 100% 313 864 HRTAP63 780698 150 WUblastx.64 (Q9Y3C9) CGI-127PROTEIN. Q9Y3C9 100% 498 860 HSLHX15 777861 153 WUblastx.64 catalase (EC1.11.1.6) - Campylobacter jejuni pir|I40767|I40767  86% 162 76 HSNBM34635131 154 WUblastx.64 acyl-CoA dehydrogenase (EC 1.3.99.-) very-long-pir|S54183|S54183  84% 1548 1979 chain specific - human 100% 251 1546HSSEF77 658725 155 WUblastx.64 (O95637) WW DOMAIN BINDING PROTEIN-1.O95637  42% 10 246  83% 296 829 HT5GR59 801930 158 WUblastx.64 (O60496)DOCKING PROTEIN. O60496  72% 70 1284 HTAEI78 637684 159 WUblastx.64(Q9UKQ2) ADAM 28 PRECURSOR (EC 3.4.24.-) AD28_HUMAN  90% 85 174 (ADISINTEGRIN AND HTEAG62 812332 160 WUblastx.64 (Q9Y5Z7) HOST CELL FACTOR2. Q9Y5Z7  60% 1 57  93% 14 2011  30% 107 631 HTEDS12 838621 162WUblastx.64 (Q9H0K0) HYPOTHETICAL 81.8 KDA Q9H0K0  97% 1029 1391PROTEIN.  42% 1269 1490 100% 16 1011 HTEHA56 806461 163 WUblastx.64(Q9H9A0) CDNA FLJ12895 FIS, CLONE Q9H9A0  94% 2 217 NT2RP2004187, WEAKLYSIMILAR TO ZIN  65% 70 468 HTEJD29 695798 164 WUblastx.64 (Q60713)REVERSE TRANSCRIPTASE. Q60713  42% 1115 1285  47% 874 1089 HTENR63877952 165 WUblastx.64 (Q9HD71) HYPOTHETICAL NUCLEAR Q9HD71  33% 12781358 FACTOR SBBI22.  78% 26 1168 HTGGM44 842856 166 WUblastx.64 probablephosphodiesterase I (EC 3.1.4.1) - human pir|T43461|T43461 100% 14001924 (fragment)  83% 1925 2488 HTLBT80 840045 168 WUblastx.64 (Q9NQQ7)BA39402.1 (CGI-15 PROTEIN). Q9NQQ7  76% 1214 1405  74% 804 1223  47% 780845  78% 313 825 HTLFA13 535937 170 WUblastx.64 (Q9UHT1) PRO1902PROTEIN. Q9UHT1  57% 1118 873 HTLGI89 835069 171 WUblastx.64 (Q9BXS5)CLATHRIN-ASSOCIATED PROTEIN Q9BXS5  98% 104 682 AP47.  99% 675 1370HTNBKL3 831967 172 WUblastx.64 (Q9Y3M2) HYPOTHETICAL 14.5 KDA Q9Y3M2 81% 123 500 PROTEIN. HTOAM11 664508 173 WUblastx.64 (Q9H5R3) CDNA:FLJ23147 FIS, CLONE Q9H5R3  77% 428 363 LNG09295.  75% 586 425 HTOHO21732808 174 WUblastx.64 P47 LBC oncogene - human pir|I38434|I38434  97%581 438 HTPCO75 853645 175 WUblasLx.64 (O00549) ORF2-LIKE PROTEIN(FRAGMENT). O00549  43% 325 26  36% 1318 1253 HTSFJ32 637720 176WUblastx.64 (Q9WUW2) VESICLE ASSOCIATED Q9WUW2  64% 747 788 MEMBRANEPROTEIN 2B.  94% 448 609 HTTCB60 853401 177 WUblastx.64 (Q9HAW0) RNAPOLYMERASE III Q9HAW0  90% 6 881 TRANSCRIPTION INITIATION FACTOR BRFU.HTTEE41 840950 178 WUblastx.64 (P78371) T-COMPLEX PROTEIN 1, BETATCPB_HUMAN  98% 92 1696 SUBUNIT (TCP-1-BETA) (CC HTWEH94 561680 179WUblastx.64 (Q9GMX5) HYPOTHETICAL 12.9 KDA Q9GMX5  60% 1150 929 PROTEIN.HTXFA72 853410 180 WUblastx.64 (Q9N083) UNNAMED PORTEIN PRODUCT. Q9N083 59% 1688 1557  66% 1839 1681 HTXKF95 834438 181 WUblastx.64 (AAH08360)Similar to hypothetical protein AAH08360  85% 233 553 FLJ22376 100% 2112 HUSCJ14 894699 183 WUblastx.64 tex261 protein - mousepir|S47481|S47481  99% 74 661 HUVDJ48 564853 184 WUblastx.64 SHORTISOFORM OF Q9P2N4 sp_vs|Q9P2N4-  92% 1510 1668 01|Q9P2N4 HBDAB91 864374185 WUblastx.64 (O00370) PUTATIVE P150. O00370  40% 907 833  35% 849 307HBDAB91 789532 193 WUblastx.64 (O00370) PUTATIVE P150. O00370  40% 587513  34% 529 5 HILCA24 869856 187 WUblastx.64 (Q9NUU6) CDNA FLJ11127FIS, CLONE Q9NUU6  95% 104 1171 PLACE1006225. HILCA24 782450 195WUblastx.64 (Q9NUU6) CDNA FLJ11127 FIS, CLONE Q9NUU6  73% 103 159PLACE1006225. 100% 168 1169 HYABC84 865064 188 WUblastx.64 (Q9H429)DJ756N5.2 (A NOVEL PROTEIN Q9H429  92% 163 618 (DKFZP727M231) SIMILAR TOTRP4-AS HYABC84 789854 196 WUblastx.64 (Q99L03) SIMILAR TOTRP4-ASSOCIATED Q99L03  89% 209 553 PROTEIN TAP1 (FRAGMENT). HMCAZ04668249 189 WUblastx.64 (Q9UQM8) CGI-44 PROTEIN. Q9UQM8 100% 9 1055HMCAZ04 887445 197 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5100% 107 1456 PROTEIN. HMCAZ04 867910 198 WUblastx.64 (Q9Y6N5)HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 106 1455 PROTEIN. HMCAZ04 858210 199WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5 100% 106 1455 PROTEIN.HMCAZ04 839783 200 WUblastx.64 (Q9Y6N5) HYPOTHETICAL 50.0 KDA Q9Y6N5100% 106 1455 PROTEIN. HE8FD92 901142 190 WUblastx.64 (Q9UJI9)HYPOTHETICAL 105.9 KDA Q9UJI9  76% 31 480 PROTEIN.RACE Protocol For Recovery of Full-Length Genes

Partial cDNA clones can be made full-length by utilizing the rapidamplification of cDNA ends (RACE) procedure described in Frohman, M. A.,et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clonemissing either the 5′ or 3′ end can be reconstructed to include theabsent base pairs extending to the translational start or stop codon,respectively. In some cases, cDNAs are missing the start codon oftranslation, therefor. The following briefly describes a modification ofthis original 5′ RACE procedure. Poly A+ or total RNA is reversetranscribed with Superscript II (Gibco/BRL) and an antisense orcomplementary primer specific to the cDNA sequence. The primer isremoved from the reaction with a Microcon Concentrator (Amicon). Thefirst-strand cDNA is then tailed with dATP and terminal deoxynucleotidetransferase (Gibco/BRL). Thus, an anchor sequence is produced which isneeded for PCR amplification. The second strand is synthesized from thedA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), anoligo-dT primer containing three adjacent restriction sites (XhoI, SalIand ClaI) at the 5′ end and a primer containing just these restrictionsites. This double-stranded cDNA is PCR amplified for 40 cycles with thesame primers as well as a nested cDNA-specific antisense primer. The PCRproducts are size-separated on an ethidium bromide-agarose gel and theregion of gel containing cDNA products the predicted size of missingprotein-coding DNA is removed. cDNA is purified from the agarose withthe Magic PCR Prep kit (Promega), restriction digested with XhoI orSalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) atXhoI and EcoRV sites. This DNA is transformed into bacteria and theplasmid clones sequenced to identify the correct protein-coding inserts.Correct 5′ ends are confirmed by comparing this sequence with theputatively identified homologue and overlap with the partial cDNA clone.Similar methods known in the art and/or commercial kits are used toamplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase.Similar reagents and methods to those above are supplied in kit formfrom Gibco/BRL for both 5′ and 3′ RACE for recovery of full lengthgenes. A second kit is available from Clontech which is a modificationof a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32 (1991). The major differences in procedure are that the RNAis alkaline hydrolyzed after reverse transcription and RNA ligase isused to join a restriction site-containing anchor primer to thefirst-strand cDNA. This obviates the necessity for the dA-tailingreaction which results in a polyT stretch that is difficult to sequencepast.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double-stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antiqense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol For Generating The 5′ or 3′ End Sequences To ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA plasmid. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′ RACE. While the full lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′ RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNA transcriptand a primer set containing a primer specific to the ligated RNAoligonucleotide and a primer specific to a known sequence of the gene ofinterest, is used to PCR amplify the 5′ portion of the desired fulllength gene which may then be sequenced and used to generate the fulllength gene. This method starts with total RNA isolated from the desiredsource, poly A RNA may be used but is not a prerequisite for thisprocedure. The RNA preparation may then be treated with phosphatase ifnecessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of the gene of interest. The resultantproduct is then sequenced and analyzed to confirm that the 5′ endsequence belongs to the relevant gene.

The present invention also relates to vectors or plasmids which includesuch DNA sequences, as well as the use of the DNA sequences. Thematerial deposited with the ATCC (e.g., as described in columns 2 and 3of Table 1A, and/or as set forth in Table 1B, Table 6, or Table 7) is amixture of cDNA clones derived from a variety of human tissue and clonedin either a plasmid vector or a phage vector, as described, for example,in Table 1A and Table 7. These deposits are referred to as “thedeposits” herein. The tissues from which some of the clones were derivedare listed in Table 7, and the vector in which the corresponding cDNA iscontained is also indicated in Table 7. The deposited material includescDNA clones corresponding to SEQ ID NO:X described, for example, inTable 1A and/or Table 1B (ATCC Deposit No:Z). A clone which isisolatable from the ATCC Deposits by use of a sequence listed as SEQ IDNO:X, may include the entire coding region of a human gene or in othercases such clone may include a substantial portion of the coding regionof a human gene. Furthermore, although the sequence listing may in someinstances list only a portion of the DNA sequence in a clone included inthe ATCC Deposits, it is well within the ability of one skilled in theart to sequence the DNA included in a clone contained in the ATCCDeposits by use of a sequence (or portion thereof) described in, forexample Tables 1A and/or Table 1B or Table 2, by procedures hereinafterfurther described, and others apparent to those skilled in the art.

Also provided in Table 1A and Table 7 is the name of the vector whichcontains the cDNA clone. Each vector is routinely used in the art. Thefollowing additional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Phagemid pBS may be excised fromthe Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excisedfrom the Zap Express vector. Both phagemids may be transformed into E.coli strain XL-1 Blue, also available from Stratagene.

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, wereobtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md.20897. All Sport vectors contain an ampicillin resistance gene and maybe transformed into E. coli strain DH10B, also available from LifeTechnologies. See, for instance, Gruber, C. E., et al., Focus15:59-(1993). Vector lafmid BA (Bento Soares, Columbia University, NewYork, N.Y.) contains an ampicillin resistance gene and can betransformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which isavailable from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008,contains an ampicillin resistance gene and may be transformed into E.coli strain DH10B, available from Life Technologies. See, for instance,Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, and/or the deposited clone (ATCC Deposit No:Z). Thecorresponding gene can be isolated in accordance with known methodsusing the sequence information disclosed herein. Such methods includepreparing probes or primers from the disclosed sequence and identifyingor amplifying the corresponding gene from appropriate sources of genomicmaterial

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X or the complement thereof, polypeptidesencoded by genes corresponding to SEQ ID NO:X or the complement thereof,and/or the cDNA contained in ATCC Deposit No:Z, using information fromthe sequences disclosed herein or the clones deposited with the ATCC.For example, allelic variants and/or species homologs may be isolatedand identified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the secreted protein, includingthe mature form, or may be a part of a larger protein, such as a fusionprotein (see below). It is often advantageous to include an additionalamino acid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification, such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the polypeptides of the present invention inmethods which are well known in the art.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:X,and/or the cDNA sequence contained in ATCC Deposit No:Z. The presentinvention also provides a polypeptide comprising, or alternatively,consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptideencoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded bythe cDNA contained in ATCC Deposit No:Z, and/or the polypeptide sequenceencoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6of Table 1C. Polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y, apolypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNAcontained in ATCC Deposit No:Z, and/or a polypeptide sequence encoded bya nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1Care also encompassed by the invention. The present invention furtherencompasses a polynucleotide comprising, or alternatively consisting of,the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleicacid sequence encoding a polypeptide encoded by the complement of thenucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in ATCCDeposit No:Z.

Moreover, representative examples of polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in Table 1Ccolumn 6, or any combination thereof. Additional, representativeexamples of polynucleotides of the invention comprise, or alternativelyconsist of, one, two, three, four, five, six, seven, eight, nine, ten,or more of the complementary strand(s) of the sequences delineated inTable 1C column 6, or any combination thereof. In further embodiments,the above-described polynucleotides of the invention comprise, oralternatively consist of, sequences delineated in Table 1C, column 6,and have a nucleic acid sequence which is different from that of the BACfragment having the sequence disclosed in SEQ ID NO:B (see Table 1C,column 5). In additional embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in Table 1C, column 6, and have a nucleic acidsequence which is different from that published for the BAC cloneidentified as BAC ID NO:A (see Table 1C, column 4). In additionalembodiments, the above-described polynucleotides of the inventioncomprise, or alternatively consist of, sequences delineated in Table 1C,column 6, and have a nucleic acid sequence which is different from thatcontained in the BAC clone identified as BAC ID NO:A (see Table 1C,column 4). Polypeptides encoded by these polynucleotides, otherpolynucleotides that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention. Additionally,fragments and variants of the above-described polynucleotides andpolypeptides are also encompassed by the invention.

Further, representative examples of polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in column 6of Table 1C which correspond to the same Clone ID (see Table 1C, column1), or any combination thereof. Additional, representative examples ofpolynucleotides of the invention comprise, or alternatively consist of,one, two, three, four, five, six, seven, eight, nine, ten, or more ofthe complementary strand(s) of the sequences delineated in column 6 ofTable 1C which correspond to the same Clone ID (see Table 1C, column 1),or any combination thereof. In further embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in column 6 of Table 1C which correspond to thesame Clone ID (see Table 1C, column 1) and have a nucleic acid sequencewhich is different from that of the BAC fragment having the sequencedisclosed in SEQ ID NO:B (see Table 1C, column 5). In additionalembodiments, the above-described polynucleotides of the inventioncomprise, or alternatively consist of, sequences delineated in column 6of Table 1C which correspond to the same Clone ID (see Table 1C,column 1) and have a nucleic acid sequence which is different from thatpublished for the BAC clone identified as BAC ID NO:A (see Table 1C,column 4). In additional embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in column 6 of Table 1C which correspond to thesame Clone ID (see Table 1C, column 1) and have a nucleic acid sequencewhich is different from that contained in the BAC clone identified asBAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by thesepolynucleotides, other polynucleotides that encode these polypeptides,and antibodies that bind these polypeptides are also encompassed by theinvention. Additionally, fragments and variants of the above-describedpolynucleotides and polypeptides are also encompassed by the invention.

Further, representative examples of polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in column 6of Table 1C which correspond to the same contig sequence identifier SEQID NO:X (see Table 1C, column 2), or any combination thereof.Additional, representative examples of polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the complementary strand(s) of thesequences delineated in column 6 of Table 1C which correspond to thesame contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), orany combination thereof. In further embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in column 6 of Table 1C which correspond to thesame contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) andhave a nucleic acid sequence which is different from that of the BACfragment having the sequence disclosed in SEQ ID NO:B (see Table 1C,column 5). In additional embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in column 6 of Table 1C which correspond to thesame contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) andhave a nucleic acid sequence which is different from that published forthe BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Inadditional embodiments, the above-described polynucleotides of theinvention comprise, or alternatively consist of, sequences delineated incolumn 6 of Table 1C which correspond to the same contig sequenceidentifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acidsequence which is different from that contained in the BAC cloneidentified as BAC ID NO:A (See Table 1C, column 4). Polypeptides encodedby these polynucleotides, other polynucleotides that encode thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides and polypeptides are alsoencompassed by the invention.

Moreover, representative examples of polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in the samerow of Table 1C column 6, or any combination thereof. Additional,representative examples of polynucleotides of the invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, or more of the complementary strand(s) of thesequences delineated in the same row of Table 1C column 6, or anycombination thereof. In preferred embodiments, the polynucleotides ofthe invention comprise, or alternatively consist of, one, two, three,four, five, six, seven, eight, nine, ten, or more of the complementarystrand(s) of the sequences delineated in the same row of Table 1C column6, wherein sequentially delineated sequences in the table (i.e.corresponding to those exons located closest to each other) are directlycontiguous in a 5′ to 3′ orientation. In further embodiments,above-described polynucleotides of the invention comprise, oralternatively consist of, sequences delineated in the same row of Table1C, column 6, and have a nucleic acid sequence which is different fromthat of the BAC fragment having the sequence disclosed in SEQ ID NO:B(see Table 1C, column 5). In additional embodiments, the above-describedpolynucleotides of the invention comprise, or alternatively consist of,sequences delineated in the same row of Table 1C, column 6, and have anucleic acid sequence which is different from that published for the BACclone identified as BAC ID NO:A (see Table 1C, column 4). In additionalembodiments, the above-described polynucleotides of the inventioncomprise, or alternatively consist of, sequences delineated in the samerow of Table 1C, column 6, and have a nucleic acid sequence which isdifferent from that contained in the BAC clone identified as BAC ID NO:A(see Table 1C, column 4). Polypeptides encoded by these polynucleotides,other polynucleotides that encode these polypeptides, and antibodiesthat bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in column 6of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., asdefined in Table 1C, column 2) or fragments or variants thereof.Polypeptides encoded by these polynucleotides, other polynucleotidesthat encode these polypeptides, and antibodies that bind thesepolypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in column 6of Table 1C which correspond to the same Clone ID (see Table 1C, column1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined inTable 1A, Table 1B, or Table 1C) or fragments or variants thereof. Inpreferred embodiments, the delineated sequence(s) and polynucleotidesequence of SEQ ID NO:X correspond to the same Clone ID. Polypeptidesencoded by these polynucleotides, other polynucleotides that encodethese polypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention.

In further specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more of the sequences delineated in the samerow of column 6 of Table 1C, and the polynucleotide sequence of SEQ IDNO:X (e.g., as defined in Table 1A, Table 1B, or Table 1C) or fragmentsor variants thereof. In preferred embodiments, the delineatedsequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to thesame row of column 6 of Table 1C. Polypeptides encoded by thesepolynucleotides, other polynucleotides that encode these polypeptides,and antibodies that bind these polypeptides are also encompassed by theinvention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of a polynucleotide sequence in whichthe 3′ 10 polynucleotides of one of the sequences delineated in column 6of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:Xare directly contiguous. Nucleic acids which hybridize to the complementof these 20 contiguous polynucleotides under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention. Polypeptides encoded by thesepolynucleotides and/or nucleic acids, other polynucleotides and/ornucleic acids that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention. Additionally,fragments and variants of the above-described polynucleotides, nucleicacids, and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, a polynucleotide sequence inwhich the 3′ 10 polynucleotides of one of the sequences delineated incolumn 6 of Table 1C and the 5′ 10 polynucleotides of a fragment orvariant of the sequence of SEQ ID NO:X are directly contiguous Nucleicacids which hybridize to the complement of these 20 contiguouspolynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention. Polypeptides encoded by these polynucleotides and/ornucleic acids, other polynucleotides and/or nucleic acids encoding thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides arealso encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence in which the 3′ 10polynucleotides of the sequence of SEQ ID NO:X and the 5′ 10polynucleotides of the sequence of one of the sequences delineated incolumn 6 of Table 1C are directly contiguous. Nucleic acids whichhybridize to the complement of these 20 contiguous polynucleotides understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention.Polypeptides encoded by these polynucleotides and/or nucleic acids,other polynucleotides and/or nucleic acids encoding these polypeptides,and antibodies that bind these polypeptides are also encompassed by theinvention. Additionally, fragments and variants of the above-describedpolynucleotides, nucleic acids, and polypeptides are also encompassed bythe invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence in which the 3′ 10polynucleotides of a fragment or variant of the sequence of SEQ ID NO:Xand the 5′ 10 polynucleotides of the sequence of one of the sequencesdelineated in column 6 of Table 1C are directly contiguous. Nucleicacids which hybridize to the complement of these 20 contiguouspolynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention. Polypeptides encoded by these polynucleotides and/ornucleic acids, other polynucleotides and/or nucleic acids encoding thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides,are also encompassed by the invention.

In further specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, a polynucleotide sequence inwhich the 3′ 10 polynucleotides of one of the sequences delineated incolumn 6 of Table 1C and the 5′ 10 polynucleotides of another sequencein column 6 are directly contiguous. Nucleic acids which hybridize tothe complement of these 20 contiguous polynucleotides under stringenthybridization conditions or alternatively, under lower stringencyconditions, are also encompassed by the invention. Polypeptides encodedby these polynucleotides and/or nucleic acids, other polynucleotidesand/or nucleic acids encoding these polypeptides, and antibodies thatbind these polypeptides are also encompassed by the invention.Additionally, fragments and variants of the above-describedpolynucleotides, nucleic acids, and polypeptides are also encompassed bythe invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence in which the 3′ 10polynucleotides of one of the sequences delineated in column 6 of Table1C and the 5′ 10 polynucleotides of another sequence in column 6corresponding to the same Clone IQ (see Table 1C, column 1) are directlycontiguous. Nucleic acids which hybridize to the complement of these 20lower stringency conditions, are also encompassed by the invention.Polypeptides encoded by these polynucleotides and/or nucleic acids,other polynucleotides and/or nucleic acids encoding these polypeptides,and antibodies that bind these polypeptides are also encompassed by theinvention. Additionally, fragments and variants of the above-describedpolynucleotides, nucleic acids, and polypeptides are also encompassed bythe invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence in which the 3′ 10polynucleotides of one sequence in column 6 corresponding to the samecontig sequence identifier SEQ ID NO:X (see Table 1C, column 2) aredirectly contiguous. Nucleic acids which hybridize to the complement ofthese 20 contiguous polynucleotides under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention. Polypeptides encoded by thesepolynucleotides and/or nucleic acids, other polynucleotides and/ornucleic acids encoding these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention. Additionally,fragments and variants of the above-described polynucleotides, nucleicacids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of a polynucleotide sequence in which the 3′ 10polynucleotides of one of the sequences delineated in column 6 of Table1C and the 5′ 10 polynucleotides of another sequence in column 6corresponding to the same row are directly contiguous. In preferredembodiments, the 3′ 10 polynucleotides of one of the sequencesdelineated in column 6 of Table 1C is directly contiguous with the 5′ 10polynucleotides of the next sequential exon delineated in Table 1C,column 6. Nucleic acids which hybridize to the complement of these 20contiguous polynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention. Polypeptides encoded by these polynucleotides and/ornucleic acids, other polynucleotides and/or nucleic acids encoding thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides arealso encompassed by the invention.

Table 3: Many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases and mayhave been publicly available prior to conception of the presentinvention. Preferably, such related polynucleotides are specificallyexcluded from the scope of the present invention. Accordingly, for eachcontig sequence (SEQ ID NO:X) listed in the fifth column of Table 1Aand/or the fourth column of Table 1B, preferably excluded are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 and the finalnucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the finalnucleotide of SEQ ID NO:X, where both a and b correspond to thepositions of nucleotide residues shown in SEQ ID NO:X, and where b isgreater than or equal to a +14. More specifically, preferably excludedare one or more polynucleotides comprising a nucleotide sequencedescribed by the general formula of a-b, where a and b are integers asdefined in columns 4 and 5, respectively, of Table 3. In specificembodiments, the polynucleotides of the invention do not consist of atleast one, two, three, four, five, ten, or more of the specificpolynucleotide sequences referenced by the Genbank Accession No. asdisclosed in column 6 of Table 3 (including for example, publishedsequence in connection with a particular BAC clone). In furtherembodiments, preferably excluded from the invention are the specificpolynucleotide sequence(s) contained in the clones corresponding to atleast one, two, three, four, five, ten, or more of the availablematerial having the accession numbers identified in the sixth column ofthis Table (including for example, the actual sequence contained in anidentified BAC clone). In no way is this listing meant to encompass allof the sequences which may be excluded by the general formula, it isjust a representative example. All references available through theseaccessions are hereby incorporated by reference in their entirety. TABLE3 SEQ ID cDNA NO: Contig EST Disclaimer Clone ID X ID: Range of a Rangeof b Accession Numbers H6EDM64 11 841331 1-2596 15-2610 AL529288,AL514648, AL523579, AL523918, AL530571, AL528848, AL523917, AL523578,BE795355, BE614208, AL529287, BE797988, BE747962, BE798201, AL530750,BF689293, BE884814, BF508994, BE798313, BE613450, BE787266, AW131835,AL530749, BG248495, BE386285, BF526775, BE873469, BE299650, AL042569,BE621187, BG168950, AW410458, BE883794, BE869375, BF348689, AW239351,BE737181, BE734276, BF309636, BF129214, BG180549, AW410610, AW601905,BE621858, AA689552, BF310547, AW960649, BF953086, AL045821, BE882424,BF724804, BE019151, AW246108, BG179779, AW374338, AW675186, BE279317,BG011956, A1475847, A1394166, A1142042, AW068652, A1539419, A1970048,A1792316, AA536006, AW272491, BG012645, A1827847, BG254459, A1673493,AW007399, A1719374, AA994188, BG176564, A1707847, AW104963, A1220974,AA022523, BF807054, BG012634, BF803094, N24911, AW665019, A1458806,AA689495, AA480131, AI808412, N41812, W17347, BE772562, BG012642,BF807055, BE772573, BE011957, BE012641, AL514647, F22287, A1160580,A1149344, BE772556, AI870582, BE772568, AW801577, BG176616, AW801325,AW068651, A1197831, BE265961, AA483525, BE772566, BE772574, BE693737,AA687509, BE839398, BF799200, AA687451, A1201450, BF896481, BE772569,BE244158, BE772576, BE826728, A1452812, BE772561, AA317941, AA308425,AA745895, AW751437, BG256219, AA782657, BF373198, AA364848, AL039960,AA405870, AW963550, BE300303, N78953, AA112404, BE826586, A1061434,AI143698, AW087863, AI382254, AW731818, BE788591, AW304748, AI589259,AA357514, BF663656, AW673017, AW664622, AA524482, AW246627, BE831243,BE831271, BG055766, AI749023, AA380438, BF746714, BE839346, AW084279,AAI13160, BF529848, AI160508, BF764174, BF752908, AA053148, AW842671,F32117, AI190107, BF752929, BE547478, AA977756, AA360528, AA022454,BF808843, BF813892, AI917965, BG011699, BG012316, BF373193, BG122581,AA622680, BF688484, BE772558, AA053706, AA733114, BG012318, AW880294,AA482098, BE256450, BE831281, BF765811, BF803085, BE243388, AA774840,AA576098, BE831236, BE772816, AF024631,2, BC007198,1, BC009285.1,AF096303.1, U73627.1, AF061779.1, AC004923.2, AF238378.3, AC000385.1,H6EEU40 12 757048 1-937 15-951 AL534759, AL521087, AL523775, AL518427,AL518354, AL517326, BE741563, BF569745, BF337372, BF570471, BF969174,BG032740, AL536265, BF026597, BE274743, BE546314, AL522079, BE538554,AW960892, BE395781, BE465235, BG167967, BE275462, AI160737, BF804270,BE538514, AA653290, BG163271, AI341701, W94467, AW084148, AI634272,AI634641, AI266283, AI366893, AW409760, AW075307, BF223869, AA604286,AI262840, AA479733, AI985719, W94359, AI271832, AW439127, AI740653,AI500535, AI674680, AW245294, BF032823, BE350203, AI869835, R85540,BF437722, AI394604, AA825592, AW469385, R60570, AI620873, AW470050,AA788601, BE300983, R72347, N77923, AA939017, R72299, R87968, R85120,Z41206, T16501, AA482633, H40664, AA297447, AA081389, R85549, AA558602,AW175922, AA987713, AI628307, AI199953, B0056252, AI382799, AA968853,AAI48651, AI289139, AI281228, AA298765, F09249, W94940, AI282067,AI262509, AI200241, AV735503, AI921784, AI628244, T32046, AA302912,AI417848, AI468747, AW055372, AA857797, AA244103, AI581120, AA679586,AI634273, AA694158, AA946762, AA608791, AI124016, H46898, W46963,AA584396, AI797302, AL518355, AL523776, H27881, AL518428, H84434,N99004, AI540357, D31570, BF765616, AAI32303, AA814926, BE812370,AW404688, AL532367, AL534760, AA887999, AL530884, AL526404, BE937700,R46056, AL517327, AL536266, R40219, AW468110, H24286, AA522908,AI567331, H26975, T81176, AW369400, T80775, AI952287, AW797699,BE782422, AA872110, BE613072, AW269694, BE870596, AA490287, AI336931,BE937686, BE741460, BE937697, AI910098, AI583322, BE962616, BE932414,AL533205, AI905196, AL521088, AA479862, AK000120.1, AL096714.1,BC007519.1, HACAB68 13 584773 1-1286 15-1300 BF967733, BF340072,AW058572, BE877116, BF029667, BE221318, BE042897, BF434234, BE966145,BF593609, AW966641, BE549675, AI692588, BF433926, W68167, AW674743,W67708, BG163487, AI802057, AW051536, AW005086, BE073104, AU145008,AI634647, AI743810, N51396, BE218196, AI857811, AI816124, AI802067,AI095027, BE503637, BF669349, AI925492, BE669954, AI813855, AI811403,BG236435, AA833834, BE073105, AA748470, AW975666, BE502705, N56917,AI146547, AI949209, AI492350, AI190896, BE219670, AI167132, AW013890,AI089941, AI810922, AI804940, AI689151, BF699838, AW873589, AA209320,N62725, AI420094, AI221693, BF130415, AI301467, AA808217, AW5I1885,BE073003, AW166094, AA019916, AI359094, BE073109, AI753256, AW675323,BF671156, AA258518, AA954483, AA324329, BF668455, F13496, AA281446,BF247796, AA487161, BF029971, AA730575, AAI21642, AI123192, R49582,AI887042, AA487312, AA364288, AA385769, AW440846, R60975, AW451535,AA972339, AI091153, T74984, R36295, AW118180, R75731, BE003024,AI459209, BG055090, AW895451, 1180344, AI984894, AA581815, BF877111,AW805837, BE000523, D57701, T03076, AI767454, F10499, Z40296, R43580,AA081798, R49916, BE928534, Z43703, AA493265, AA526871, AU118452,AI144481, BE540542, AL442081.1, AL354793.11, AK001029.1, AF189009.1,AB015344.1, AF293385.1, HACBJ56 14 847112 1-874 15-888 AAI57001,BE348653, AW027639, AA534339, AW001883, AA363258, AW959379, T71037,AW953765, BE048583, BF878388, T67200, AW393348, AW393350, AW384705,AW386713, AAI56760, AW055343, BF892732, BE140594, HADMB15 15 8471161-316 15-330 AW136268, BG056888, AI131328, AIl74443, AI091646, AW117296,AW168872, AI082447, AI432175, AI290911, AI741489, AI682685, AI142536,BG059892, AW149659, AW071935, AA233541, AI183690, BG056462, AI689641,AA599916, BF196591, BF196843, AAI99743, AW136277, N77910, AA564806,AA243035, AA779709, AV722133, AI032138, AA844525, AI467910, AW965361,AA852418, AI982751, AI282445, AI982761, T03902, AI420648, AW167499,H08108, BE328548, AW068986, C15651, D52660, AW665899, AI246702,AI538705, AI271662, AI435112, AI288692, BE466948, AI690048, D55112,AA779042, AL536118, D53747, D54101, AA486941, D53384, W07076, AA232504,AA486765, BF832290, AI038647, AW497637, BF947006, AU155428, T05461,AL136582.1, BC001207.1, AB040527.1, AB058762.1, AB040528.1, AB040529.1,HAGDW20 16 637489 1-1270 15-1284 AI671549, AA603387, AA614197,AC005629,2, AC006453.3, AC020898,5, AP001610.1, AC025435.5, HAGEQ79 17828055 1-771 15-785 AI741487, AA779582, BE674646, AW303577, AI305251,BE219521, AI688718, AI936253, AI093754, AW341275, BE222507, AI692909,BF966664, AI493111, BF525487, AW016639, W92767, AU150022, AW341787,AI278427, BF966817, AA044775, AA910036, AI685015, AI285959, AA719683,BE645673, AW196910, AI432636, AI096735, BE618873, BES41159, H91757,AI702190, BG152855, AI244929, AI681847, BE217959, AI498036, BE467879,BE696146, AI810609, R55798, AA897359, BE464034, AA975324, F09971,W73069, AW594097, AA703815, BF224038, F10760, T72606, AA932659, H46138,AA351671, T31206, AA317283, AI867144, AI681277, AV718692, BE938093,AV718489, T31188, AI874073, AV724520, D80253, D80219, AW582752, D80240,D80210, AV719783, D80134, D59275, AV742001, D80391, D51423, AV720464,AV718844, AV720731, D80227, D51250, AV701332, AV699550, D59619, D80193,AV722801, AV699447, AV720220, AW973447, AV742667, AI306486, AV743601,AV745080, AV701443, AV744934, D80949, AV701017, AV742720, AI305252,AV741012, AV701043, AV701248, AV700229, AV743008, D80196, D59927,AV701428, AV719000, AV701004, AV745831, AV744773,D80043, AV701125,AV701154, AV719913, AV699927, AV701431, AV721784, AV700889, AV744771,D80366, M79008, D59787, AV744768, D81026, AV723927, AV701261, AV720203,AV745724, AV720812, AV740535, AV745723, C14227, AV720607, C14014,AV701012, AV701149, AV701118, AV737584, AV745366, AV701163, AV745369,BE702445, AV723097, AV70L166, BE702442, AV744770, AV701422, D50995,AV701344, AV699479, AV699669, AV719822, T11051, AV699866, AV701055,BF224150, AV701227, C75259, D80045, D80168, AV699746, AV746385,AV719324, AV701145, AV718707, AV745488, AV701021, AV701330, AV746335,AV701121, AV742671, AV700357, D80022, D80195, AV700622, AV720211,D58283, AV745392, D59889, C15076, D81030, AV701335, AV746162, D80188,D59467, AV745920, AW959570, D80038, AV701130, F13647, D51799, AV701419,AV718770, AV745847, AV701013, D80378, AV745388, T03269, AV721386,AV745490, AV718800, AV701415, AV718681, AW904616, AV700495, AW949642,AV701151, AV719468, AV738934, C14429, AW978634, AV745853, AV745197,AW949645, AV701231, AV701357, AW965158, AW975618, C14298, AW949633,AW949632, AW949641, D80212, AV699682, AV745621, AW949629, AA285331,AI557751, T11417, AW966531, AV720150, AV701224, AW949653, AW949658,D50979, AW949656, AW966062, D59502, AK022817.1, AL096677.21, AF271371.1,D34614.1, X67155.2, S78798.1, D88547.1, X92518.1, AF058696.1,AB028859.1, AB002449.1, AB035274.1, X98248.2, X60736.1, U79457.1,D50010.1, AB033111.1, AB038216.1, HAJBV67 18 866415 1-2522 15-2536BG252656, BF732416, AV713753, BE905485, BF062374, BF445098, BF110352,BG252894, BE620095, BG249923, BE867752, AW606977, BG171028, AW576585,BE868698, BF671587, AW860769, BF941584, BF986308, AW305358, BF037687,BE541890, AW958924, AW974216, BF105260, AL048954, BF434917, AA057428,AW860733, BF664978, AI040432, BF984881, BF114918, BE872774, BE349491,AW263003, BF697715, BF382321, BE938703, AI378631, BF447674, AA446149,AA044378, BG114831, BF815345, BF085497, BF815237, BF210190, AA579908,BF132467, AA437015, AW860753, AI741531, AI742016, AI963805, AV748930,AA457625, BF815346, N31845, AI927889, BF699623, AA587067, AA831367,AI038411, AA442844, AI382172, BF084350, AW993684, AW407667, BF029928,AW028681, BE327066, BF887305, AV695738, BE222425, AV696527, BF755168,BE876090, BE167030, AI768063, BE000825, H12700, AV708152, AW001069,H03274, BF063098, BE933732, BF815719, BF594797, AW974217, N93209,N23944, AI290752, BF802746, AA557778, AA604449, Z32781, BE004621,AA910221, AA226865, R78864, BF326913, BG179582, AI370350, BE719765,BE768063, BE932712, AA780882, T31498, AW798498, AI635435, BF088211,BF817478, Z28655, BF943308, Z24930, BE932705, BF126152, AI015125,BF981166, AI684725, T36185, H12701, R37535, AW952059, AI689130,AA296931, AW798657, AW364905, R79351, H03275, BE768230, AW206046,AA081583, AA936681, BG104571, BE176285, AW993023, W69607, R31681,AI479514, BE696398, AA716370, BE463676, AW366456, BE869217, BF064127,BF001446, AW884802, AW999085, BG104993, AI039088, R31723, AW366514,BE871677, AI241206, AI743907, AA306185, BF037794, D61175, AA852523,AW365573, BF799275, AAI29989, BF985004, AW838470, BF802748, R36687,BE001097, AA723997, BE932064, AW366145, BF230069, AW999007, AW993306,BF733961, AA508532, AW972441, AW972636, BE932056, BE086739, AAI64808,BE064535, BF986296, BF095055, AW408116, BE184804, BE184805, BE184738,BE695142, BE184803, BE172976, BE184743, BF984676, BE184732, BF741954,AI366900, AW082623, AW118518, AI698391, BF871314, AI954504, BF753053,AI679312, BE967260, BF207979, AL515195, AW050850, AW089844, AW151136,AL515191, BE965599, AI619607, AI687568, AI540674, AI345688, AL513817,AI590043, BG032036, AA806028, AL043168, AA329665, AI923989, AI670002,AA641818, AI866770, AI679321, AI591420, AI473451, AI445165, AW051088,BF812961, AL514093, AI521560, AI633125, AL514871, AI915291, AW152182,AI247082, AI582932, AI889189, AI587121, BE875959, AA743012, BF814412,AW193894, AL515413, BE911554, AI918449, AF269150.1, AK027788.1,AK000756.1, AF116347,1, AK027438.1, AF160213.1, AF124819.1, BC009311.1,BC001967.1, AB048975.1, AL137478.1, BC002733.1, AB056421.1, AL133560.1,AK027129.1, U42766.1, AL080118.1, BC001969.1, AK026927.1, U38847.1,AB047878.1, AK025857.1, BC004264.1, BC004899.1, AL137529.1, AK000323.1,BC005858.1, M92439.1, AL122100.1, BC006458.1, BC001964.1, AL353956.1,AL137557.1, AF132676.1, AL133640.1, AF061836.1, AL137533.1, AK027164.1,A3406939.1, AL049430.1, AB056427.1, AK027173.1, BC007571.1, BC003122.1,AL136784.1, AF245044.1, BC001215,1, BC004324.1, AF252872.1, AL389935.1,AL136752.1, BC003410.1, AL137560.1, BC008037.1, AL137555.1, AK026649.1,AL136767.1, AK000206.1, BC003658.1, AK026057.1, BC008284.1, AL136786.1,AL110225.1, AL133623.1, AF078844.1, AF353396.1, 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AA261835, AA262483, AA523928, AA551549, AA563835, AA857095,AA872771, AI095007, AI096629, C05812, C15709, AA247765, AA393650,AA400834, AA487693, AA488710, AA663750, Z21548, AA843596, AA844473,AI041193, AI083985, Z41640, Z46025, Z44537, F03607, D11797, AI262317,AI264408, AI304594, HCQBH72 46 637548 1-1782 15-1796 AA640538, AW974686,BE144592, AA649644, AA649707, R31618, AA652004, R32348, HCQCC96 47845066 1-2152 15-2166 BP970581, BG117166, AV695085, AV686338, AI341460,AW173384, AV693976, BF032394, B0024316, BE893802, BG254562, AW055235,W39204, BG170478, AW978735, BF572731, AW968956, AI909118, AI909124,AW592429, BG171038, AW118938, AI689438, AI419443, AI801242, AW438695,AI123971, N59864, AA707755, AA974210, AW130020, AA489046, AA768780,AI146982, AW768627, AI093766, AW889585, AW298736, BF111650, AA284319,AA907244, AW874520, AI535676, AW579265, AI955386, AA279581, AA983814,BF185409, BE622718, N59886, AI859864, AI498376, BG171039, BG116650,W01363, AI699807, AA824487, T86598, AA994605, AW044013, T85108,AA489144, AW768600, AA811658, AW271482, BE895361, AI631722, AW021293,R64514, T77523, AA736753, H44608, AI955411, AV689519, N90263, AL119283,H94626, T77559, AL119309, T86597, AI909117, AW376940, N79005, N77027,AW105078, N62828, AI701272, AI334730, T07505, AW848643, AW243861,AI909110, BG007292, BE162291, BE162293, AA532611, AP001816.2,AL022153.1, AC004804.1, AC015982.9, AC006840.17. HCQCJ56 48 8321571-1273 15-1287 AI674974, AI217307, AA813576, BE302346, AI824976,AA994749, AI244904, AI262935, AA020796, AA234517, BF432061, AA443035,AA463478, AW079079, AA694400, AI005463, AA776532, R00437, R00438,AL513043.7. HCUDD64 49 835082 1-388 15-402 BF109963, AI870761, AI149403,BE675981, AI979111, AI590348, AI769440, AA568609, F04371, R68556,N24429, R85927, AW973928, W02539, BG150863, R79201, N694 12, R79466,T80848, AI494453, R28549, AW440020, AL390151.1. HCWAE64 50 535893 1-45715-471 AL043265, BE895962, BF091850, BF924502, BF930204, AW973724,BE906549, BF972009, AA558125, BG163769, AW993087, HDPDI58 51 5872651-1983 15-1997 AW629326, AA642298, AW973571, AW293886, AA714702,AA489697, AW665911, AW119199, AA553561, AW768356, AA744636, AI806778,AA768815, AI221150, AA732540, AV713604, BE247593, T84374, AA354491,T07354, AW273936, AA648816, AI199572, AL133087.1, AC013264.4,AL117381.32, AC019105.7. HDPFU43 52 790189 1-1890 15-1904 BF983796,AV653552, BE259910, AI766649, AV703996, BF304737, BF433988, AA700236,BE178896, AI338643, AI354469, AI823774, AI379434, AI139748, AA934777,AI424295, AI280893, AI360455, AI539184, AI750403, BE184282, BF675256,AI262328, AV653641, AV687758, AV688883, AI538841, AV683306, AV686477,AV687759, AV688680, AA903947, AV689834, AW957949, AI079715, AAI28542,AI160482, AW804386, AA459614, BF770031, AW014830, BF848624, AW859834,AV685347, H94111, BF807109, AW470950, AA316165, AI400889, AA459389,BF924526, AI083688, AA374022, AW086191, R45973, W21315, R63000,BF798131, N93502, BF798175, AA304841, T39902, BF839163, C02619,AI373388, AV752433, AI750402, H94110, AI015368, AI916914, AA531491,AA215914, AW051088, BG180527, AW983783, AI470293, AV681824, AW023338,AW827289, AI929108, BF814357, AI440263, AA579232, AL040694, AI433590,AL042627, AW087445, BF871314, AW162194, AI537677, AI698391, AL037454,BF910810, AI923989, AV738730, BF904265, AV723064, AI345688, BF792445,AI921379, AV657079, AW020397, AV702021, BF814018, AI859991, BF184134,AA808175, AL043975, BE964614, AI446538, AW827118, AW150511, BE908107,BE965121, AV756990, AL514823, BE965758, BE906419, AV714036, AV682345,BE965621, AI623941, AI969655, BF750879, BG031068, BG036067, AI340519,AW452992, AW128931, BG113169, BE972047, BE538997, AL049085, BG260052,BE904851, AV717927, AL110306, AA635382, AW834221, AV706915, AI251221,AW022682, BG165323, BE965432, BE967307, BE965067, AW881086, AI340603,AI560099, AA427700, BE775251, BE965599, AI284509, AI863241, AA857847,AI866465, AI524608, AI538850, AA420722, AI918634, AL039011, BE439835,AW020048, AL080033, AI567351, AW089844, AA613907, AW163554, AI050666,T99953, BG256950, AW075084, BF885000, BG027280, BE878028, BE885490,AI349937, AI334884, AI307708, AL036187, AI963068, AI312325, BF925729,BF218049, AV761001, BG168696, BG179666, AV728833, AI671642, AI801325,AV729462, AI565172, AI307520, AV759235, BG104769, F29308, AA883351,BF966050, AI621341, BG105895, AL119836, AI269323, AI475371, AV718300,AV757781, AI564166, BE964700, AV742848, BE047833, BF835250, AW128855,AW151138, AV685436, AI950892, AI500662, AV681721, AI247193, BF812937,AL514493, BE839731, BF339322, AL120254, AI950664, BE909150, BF822127,AI345608, AI800370, BF983610, BE875407, AV757455, AV722452, BF970162,AA651819, BG260087, AI091468, AW935969, AI536685, BC001057.1,AF049891.1, AL136623.1, AI006198.1, AF061254.1, Z95115.1, AC007429.11,AL355379.5, BC007199.1, BC006412.1, AF143723.1, AL162004.1, AF217982.1,AK026480.1, BC002631.1, BC008488.1, AL133640.1, BC008025.1, AL050149.1,AL133093.2, AL110196.1, AB056809.1, AK026649.1, BC00I418.2, AL359622.1,AL136622.1, BC004905.1, AK024538.1, AL137555.1, AF227198.1, Z37987.1,BC003687.1, BC008387.1, AK024974.1, AL117435.1, BC007346.1, AK000718.1,AK026522.1, AF225424.1, AF125949.1, 578214.1, AL049938.1, AK026528.1,AL050024.1, BC001967.1, AL512733.1, AB056420.1, BC003682.1, AL137529.1,AK026592.1, AL049300.1, AK000618.1, AB048974.1, AC006313.1, AL050277.1,AB05L158.1, BC006480.1, AIB055374.1, AB047801.1, AB055315.1, AL050116.1,AF260566.1, U80742.1, AL389935.1, X72889.1, AB019565.1, AB048953.1,BC005931.1, AJ242859.1, AL133075.1, AK000310.1, AK025254.1, AL162085.1,AL080057.1, BC002481.1, BC006195.1, AL389939.1, AK026086.1, AL137557.1,AB063070.1, AL353957.1, AB063046.1, U55017.1, AF219137.1, AL512719.1,577771.1, AK000197.1, AF057300.1, AF057299.1, AL137267.1, AL110199.1,AK026534.1, AB055361.1, AK025391.1, AL031984.13, BC000066.1, AL136787.1,Y16645.1, AL390154.1, AL117626.1, AB060908.1, AL162006.1, AL080234.1,BC006525.1, AL442072.1, AL512765.1, AL137527.1, AL122121.1, BC006807.1,X66417.1, Y10080.1, AK026550.1, AL049283.1, AL137478.1, BC008899.1,BC001328.1, AL136786.1, BC002485.1, AK025339.1, BC006103.1, AK026959.1,AL122111.1, AK025092.1, BC001963.1, BC004958.1, AL117649.1, BC000317.1,BC000714.1, BC007998.1, AL023657.1, AK025708.1, AL080156.1, AL110225.1,AK024546.1, AF055917.1, AF205073.1, AL080060.1, AL117648.1, AL137283.1,BC008284.1, AK026642.1, AB063008.1, AK026741.1, AK000212.1, AK027146.1,BC007517.1, AK027160.1, AB055368.1, AL136893.1, AF061795.1, AL117457.1,AF151685.1, AL157431.1, AL133606.1, AL389982.1, AK027868.1, BC002733.1,BC001795.1, AF111112.1, AK026865.1, AK024524.1, AF026030.1, BC008781.1,AB060826.1, AK026353.1, AB060893.1, AL442083.1, AK025414.1, AK027164.1,AB048975.1, AL117460.1, AK025435.1, AB060852.1, AL136768.1, AL050143.1,AB063100.1, AL133113.1, AL136892.1, AL080129.1, AL137480.1, AL080124.1,BC001191.1, AL122110.1, BC008382.1, AL133054.1, AK027113.1, BC002752.1,AK024550.1, BC007255.1, AK026744.1, AB060863.1, AB063074.1, AB055328.1,BC001964.1, AL050155.1, AL137281.1, BC002342.1, AF090896.1, AL049276.1,BC008417.1, AF104032.1, AL133665.1, AL136540.1, BC001082.1, BC007021.1,BC003619.1, BC002643.1, AL049382.1, AL136844.1, AX025209.1, AL137648.1,BC001349.1, AB055366.1, AL049466.1, AK026927.1, AK026434.1, AL050146.1,AL117394.1, AK026855.1, AK025383.1, AK026532.1, BC008365.1, AL049464.1,BC006210.1, BC005858.1, BC002839.1, AK026164.1, AY026527.1, AL049314.1,AL110197.1, AK026762.1, U68233.1, BC002523.1, BC008506.1, AX027188.1,AF262032.1, AL133016.1, AL136864.1, AL137273.1, AL136925.1, AK026526.1,576508.1, AL136749.1, AK025410.1, BC004926.1, AK024570.1, 561953.1,AK026624.1, AB049892.1, HDPGE24 53 801947 1-2611 15-2625 BE876192,AU145980, BE839859, AW953709, AV651029, AW866434, AW866436, AW866430,AV687299, AA604920, AA604512, AI887664, AW813014, BE839866, AAI64729,AI566037, AA602341, AA602613, AA214047, BE839860, AI355441, AW855356,AA506540, AUI19708, AW855353, AI884345, AV656490, AI049591, AW853687,AW995969, AI963674, BG058784, BF681462, BE811870, AA551394, BE081412,BE672638, AV687875, BE564307, AW935217, BE811892, BE145548, BE563924,AW577107, AV704081, AA631460, AW380640, AA366464, BG012149, BF694965,AW341886, AW866268, AI537997, F29519, AI537504, AI567884, BF874935,AV659506, AW363563, AA631500, AI363970, AA669020, AI270484, H78415,BE709511, AA640505, BE178526, AI989765, AW866337, AW953693, AA484751,AA342969, BF882965, AA484783, AV659374, BE796439, AV695480, AV659391,AW024055, AV659405, AI832956, T81440, AA654981, R70506, BF852810,AA484906, AW997573, AW379425, AI932609, AA631380, AA570339, BE708328,AI597820, BF694852, BE815355, AW934969, AW902128, AV684943, AA366571,BG260565, AA632800, AW007894, AW192258, AI886084, AV764490, H82763,AWI31401, T69164, AA605054, AW573583, AA834697, AW858120, AW893702,AW573573, AW074527, AV714931, AW820698, AI679343, AA558871, BF853927,AW438596, BF883928, Z32833, AA503427, AW393438, AA610678, AA522988,AA483882, R95100, AW893701, T59151, AW965008, AA848158, BE067011,T98359, T68422, AI679520, BF935516, AA528276, BE839943, BE929829,AL120269, AV759172, H02561, AV760701, AW802714, BE541237, N21656,AI457389, BE066950, T30343, AI679960, H78215, AV700663, AW978714,AL135377, AA243867, AW151713, AW102955, BF884208, AW157616, BF846275,BG034591, BF106210, BG0I1353, AAI61288, BF883454, AC000353.27,AF001893.1, AC006121.1, AC005484.2, AP001710.1, AL590762.1, AC009961.11,AL035555.10, AL160411.25, Z83822.1, AC022402.4, AL136139.6, AC005291.1,AC007225.2, AC007021.3, AC018828.3, AC010742.4, AL391259.15, AC090943.1,AC090514.1, Z98946.15, AL450263.15, AL034372.33, AC008873.4, AF002812.3,AF224669.1, AC006030.2, AL031311.1, AIA22020.5, AL049759.10, AL117692.5,Z94801.1, AC008670.4, AL022318.2, AC008892.5, AC027124.4, AP000555.1,AP001169.1, AC018502.5, AC002378.1, AL390074.17, AC083863.2, AC066597.4,AC002091.1, AC079141.7, Z94056.1, AC005157.1, AL354720.14, AC026391.6,AP00L715.1, AC040160.4, AL139109.14, Z97054.1, AC002289.1, AC007450.1,AC007482.7, AE000658.1, AL499604.9, Z84484.1, AP001724.1, AL109865.36,AC066608.5, AC073101.7, AC007850.29, AL137139.9, AL079342.17,AC018642.6, AC007773.1, AC024163.2, AL162231.20, AC007097.4,AL035685.21, AP000352.2, AL021368.1, AF111167.2, AC007363.3, AF088219.1,AC004125.1, AC009137.6, AC018755.3, AL158828.14, AC026398.4,AL356652.19, AC005846.1, AC023510.16, AL355096.4, AC034240.4,AL049713.20, AC011742.3, AL163853.4, AC015982.9, AL138743.5, AC007907.2,AC091492.1, AC021188.6, AC018682.4, AL138878.10, AL390025.1, AC006050.1,AB026898.1, AC011236.8, AC004024.2, AC005214.1, AC002464.1, AL139113.21,AC005046.3, AP000246.1, AP000207.1, AC007563.2, AC005520.2, AL137128.4,AL031670.6, AL442167.1, AL163285.2, AC011816.17, AC024166.3, AC011739.7,AC013734.4, AL021154.1, AC005881.3, AC005697.1, AL022165.1, AL391114.12,AL023513.1, Z98044.13, AC0000.94.3, AP000129.1, AL139415.10, AC011242.8,Z98304.1, AL589693.3, AL391122.9, AL445435.11, AC003071.1, AF131216.1,AL360272.23, AC087071.2, AC003962.1, AL136123.19, AC020908.6,AP001830.4, AC008450.5, AL445669.9, AJ271736.1, AL034550.31, AL118557.5,AC008891.7, AP000782.3, AP000500.1, AC011464.5, AC0L13L1.11,AL158196.24, AC025262.27, AL049869.6, Z93341.5, AC022468.5, Z92542.2,AL034384.1, AP001728.1, AL031387.4, AC002994.2, AL591807.1, AC022407.6,AL160231.4, AC007065.5, AC005539.1, AL050318.13, U91322.1, AL133415.12,AC010252.3, AC068781.18, AL133500.3, AC008011.11, AI400877.1,AL390738.4, AC044797.5, AC006445.11, AP000688.1, AL445071.14,AL133545.10, AL035089.21, Z98884.11, AL355535.14, AC007999.12,AC022007.3, AC083871.2, AP000350.1, AL354696.1 1, U80017.1, AP001671.1,AL121997.7, AC007282.4, AL139330.17, AL049779.6, AL163282.2, U82671.3,AL445928.8, AP001412.2, AC022392.4, AJ400879.1, AL161454.10, AC090957.1,AP001922.4, AC003091.1, AC005971.5, AL121905.23, AL161935.10,AC022267.8, AL158141.14, AC025165.27, AL158198.14, AL160036.12,AL136303.15, AC004707.1, AC012450.9, AE131215.1, AL132657.33,AC006965.3, AL161937.13, AC005969.4, AC018633.2, AC004764.1,AL359846.11, AL118556.4, AF196970.1, AL137787.11, AL360169.17,AC018769.2, AC000052.16, AF190464.1, AL137100.4, AL445248.7, AC008738.6,AC018523.9, AC005670.1, AC010605.4, AC007541.9, AL049795.20,AL096712.20, AC006057.5, AC004019.20, AC006071.1, AC026166.4,AL132640.4, AC022201.4, AC008569.6, AC003049.1, Z84469, 1, HDPIU94 54813352 1-2182 15-2196 AU140297, AL529544, 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AI431312, AI432655, AI431310, AW128900, AI431238,AL045327, AI431354, AI432666, AA580821, AI431347, AI431315, BF448552,BE672748, AI432661, AL134524, AI431323, AI431337, AI432675, AI431321,AI492519, BE672745, BE672732, AI431246, BE672719, U46344, AI431235,AI431243, AI432647, AI432651, BE672738, AI431255, AI432674, AI431330,AI432649, BE672767, AI791349, AW601637, AI431248, AI431241, AL042842,AI431254, BE672774, AI431357, AL042729, AI432672, AI432665, BE672742,AL042931, BF589777, AI432662, BE672627, AW577201, AI431345, BE672644,AL042655, AI431351, AL042508, AI431231, AI431346, AL042853, AI432676,AI432673, AI432658, AW128884, AI431257, AW577199, AL042533, AL043166,AL047611, AI431340, AL135012, BE672622, BE672792, AI432657, AL042802,AW128846, AI431247, AI432664, AI432645, BE672718, AL042787, AL042515,AL042832, AI431751, AL043295, AI431314, AI492520, HDPIY31 55 8861591-1964 15-1978 AL533296, AU142272, BE560264, BG117407, BE407326,BG116397, BE314927, BE315405, BF205715, AI935180, BE313422, 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AB047615.1, AK026959.1,BC004951.1, HTGGM44 166 842856 1-3002 15-3016 AW974580, BE764084,AA651951, W01997, H68969, AI459019, H70945, AA486949, T66948, T66949,AA486772, BF329143, BF108414, AW469166, AI568694, T57664, AL133623.1,HTHBZ06 167 832477 1-609 15-623 BG107523, BG180234, BF668800, AL514985,BF339863, AI400160, AI566873, BE909457, AW262875, BE906621, AW470063,AI758577, BE907206, AA777509, AV715444, AWL31846, AA406614, AW087747,AI811951, AI371781, AI742506, AI337891, BE738291, AA934901, N40173,AW157527, AI742505, AI374781, AI081113, AW173107, AI379523, AV756830,AI139790, AAI95689, AI801399, BG054839, AA532727, AA235284, AI087379,AI792601, AI952545, AI245243, BG026067, AI805770, AA600140, AI040546,AV703045, AI753737, AA625963, AW591860, AAI59931, AA477326, AI360032,N40209, AI864174, BF197737, AA430365, AI829158, AI869836, AI955815,AI804015, BF909529, N30689, AA478600, C16344, BE906555, AI640196,AW072764, BF306291, AA905154, AA481723, AA758776, AI371005, R78607,BE783860, AA865424, 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BE782619, BF795936, BG248163, BE869185, BE893350,BF435225, BE905414, 8G026633, BF033083, BE970046, BE885230, BF692625,BF692541, BE892741, BE393898, BE389940, BG163906, BE313920, BE312030,AI741613, AI762578, AI241474, AI813813, AI922418, AI990378, AA018345,AW631237, AW151233, AI400794, AI420163, BE550276, AI949071, AW963076,AI922430, AA614565, AW051437, AA018346, AA612852, AI000311, AI338519,AI360869, AW613433, AI356485, AW590872, W56183, AA417581, AI214800,AI671156, AA451942, BE242648, AW390145, AA00L0L9, R69763, AI419907,AA768838, AA482598, AI696492, AW473585, AI674961, BE868575, AI247090,H08477, AA576510, BE391031, H06603, AI018102, T56902, W56260, BF372172,AA00L020, AI285366, R87304, BF928779, BG152437, AA814595, AI569050,AI239612, H06633, AI623626, R69764, AA971138, H07140, BE856299,AI357631, AI001995, AW243905, R48323, BF003017, H43938, H52166,AA026966, BF988194, W22979, BF372164, H89737, R87305, BF677308, R48432,AA347636, AW080561, AA593837, AI459770, T56903, H08759, 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AL512746.1, AF091084.1, AB060912.1, AL359615.1,AB019565.1, AF090901.1, AL122050.1, AL133560.1, AF090934.1, AB060826.1,AK027096.1, AL359618.1, BC008387.1, BC002733.1, AL162006.1, BC001045.1,AB060852.1, AL442082.1, AK026542.1, AL133606.1, AL049314.1, AK000053.1,AB052200.1, AL136892.1, BC006195.1, BC007021.1, AL117460.1, AK026452.1,AL359941.1, AL162083.1, AK000445.1, AK026045.1, AB060863.1, AB048954.1,AF125948.1, AK026592.1, AB051158.1, AB048953.1, AB047904.1, AL133080.1,BC008899.1, AF218014.1, AB063008.1, AK026959.1, Z82022.1, AB060908.1,AL133075.1, BC003687.1, AF097996.1, BC006807.1, AL137459.1, AJ242859.1,AL117435.1, AL049452.1, BC008417.1, AL137550.1, AF090943.1, AL050277.1,ALL10196.1, AK026741.1, AB060825.1, AL136789.1, AF090903.1, AL136799.1,AL157431.1, AB055315.1, AF078844.1, BC007199.1, AL136928.1, BC001967.1,AK025339.1, AK026608.1, AL359596.1, AL110221.1, AL117457.1, AK026534.1,AL050116.1, AL353940.1, AF090896.1, AL049464.1, AL389978.1, AK026784.1,BC008365.1, AL080124.1, 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AC010320.9,AL356915.19, AF053356.1, AF312032.1, AC020916.7, AC0I1895.4, AC020906.6,AF134726.1, AC004253.1, AC008738.6, AC020908.6, AL133387.8, AC002126.1,AC005522.2, AC005620.1, AC078818.19, AL359091.10, AC005052.2,AC004089.25, AC005071.2, AL160269.14, AC006312.8, U91321.1, AC005015.2,AL137792.11, AC004826.3, AC007374.6, HTXFA72 180 853410 1-1847 15-1861AW007854, BE677425, AW297663, AC008102.17, AC067742.5, AC012476.8,AC007637.9, AC004887.2, AC007383.4, AAL000426.3, AC020916.7, AC006538.1,AC025166.7, AL133325.20, AL121754.18, AC024075.4, AL109758.2,AAL001574.3, AL080243.21, AC002365.1, AL499604.9, AC005702.1,AL121905.23, AC008738.6, AL049832.3, AC0L1491.5, AC020744.4, AC004953.1,Z84484.1, AC006559.6, AC008440.8, AC003101.1, AL157827.17, AL133214.12,AC008946.6, AL357498.16, AC003046.3, ALI61793.9, AC011895.4, AC011442.5,AC009756.9, AL157838.24, AC004973.1, AL133448.4, AC008699.5, AL356019.5,AC002119.1, AC008537.5, AC011461.4, AC074295.7, AC037475.9, AC015842.9,AC008379.6, AC004707.1, 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AA454974, BE621103,BG249254, AW880574, BE315071, AA724393, AW401755, BF981216, BF724854,BE740318, AW602164, AAI76450, AI798066, BF529155, BF590827, BE938687,AA528038, AAI76930, BE007448, BG260090, AA744235, BE019877, AI755131,BE019722, AA258968, BE007599, AA888876, BE206323, AA454975, W49835,AI658890, AI400439, BE855719, BE965617, AI369432, AI378678, AI814475,BE894966, BE255155, N33371, H30709, AI817038, AI079528, AA988238,AI094675, BE296010, BG055258, BE698523, BF749938, AA781122, BG008471,BE007089, AI078131, BG012658, H28137, AA636058, EF112108, H30791,W52270, AI582733, AI150949, AA410493, AA860936, AAI32898, AI278541,BFL12247, BE044217, BF507690, AA933024, H66966, AA258022, BE247272,AA310300, BE208182, AA256356, H30612, H24913, N47554, AA595919,AA921999, BF772395, AW883922, AI050086, BF820414, AA326738, H66965,W52173, AW630747, AA987810, BF841176, BF841178, H24636, BF886659,AW025934, R85382, AAI76415, AW997264, BE769213, BE873578, AA213621,AA213747, AI090506, AI350930, AI335914, R88068, BE315307, AA306524,R78161, BF772700, BF755261, R25528, BE698518, AA367585, BF880127,BG163239, AW964548, R88419, BE833357, R67073, AW168072, AI866652,R66194, W39693, BF512635, AI921135, R77799, BE742426, R88069, Z25017,BE005706, T52535, AI084635, AW272406, BE005705, AA368103, AA485159,H44289, H21572, AA335920, AAI32726, BE007543, W37836, AI080447, T72649,R23331, AI800373, H11232, AI493270, AA633490, R61159, R48336, R45587,AW591739, BE315089, AA375973, AA091597, AI961784, AW140066, R61873,BF806323, BF342050, Z28722, A1003986, AW608071, T30986, H10607, R48240,AAI43657, T72717, AA378816, AW768907, H28184, AA320516, W45049,BF743131, N47553, BF115823, AA988048, AI698305, AI342699, AA410492,AAI76682, AA366872, BF432748, AW895119, AA329961, AA873382, N51170,AI802557, AW947291, BF432749, AI468036, BE315041, BE814237, H10399,AL045269, AI307370, T52614, AI310496, AW879611, AA463709, AW842163,BE245828, AI364211, AA485063, D20903, BE181425, BE281137, AW998715,AL080223.1, AC007040.2, HUVDJ48 184 564853 1-1813 15-1827 AI479925,AV720735, AI886110, AF261918.1, AB037733.1, HBDAB91 185 864374 1-99315-1007 AI167963, AA782398, BE043035, AW051006, W07319, R23362, N75825,AW195519, BE858969, AI701657, R45349, AI468816, AW858522, AW577199,AI135012, BE927373, AW601637, BF084778, AL134110, AL134524, AW577201,AL045327, AL045494, AL042523, AW577192, AL045328, BF910726, AL042420,HELGG84 186 851137 1-1095 15-1109 AI822137, AI821793, AI141174,AI742807, AW008096, AI376221, N70665, AI147430, N66810, W05747,AW894967, AI708189, AI792140, N75004, AI597655, AI140241, HILCA24 187869856 1-1968 15-1982 BE780749, AU137314, AV732875, AW954734, AW138881,BF681107, AI079555, AI624252, AA233208, AU157126, AI734898, AW088851,BE221267, AA314962, AV715966, AA971982, AA233124, AAI29416, AAI33798,AA886808, AA353195, AW132033, T98200, H50558, AI888751, AI818363,BF917932, BE926224, AI784628, H50559, T98201, AK001989.1, AL512750.1,AC010627.5, AC01049 1.3, AC026749.5, AC016656.5, AC016652.5, HYABC84 188865064 1-1464 15-1478 BE619984, BG180257, BG253753, BE546940, BE795721,BE871790, BE538846, AW953562, AA524254, AW978620, AW970777, AW001609,AI798108, AU159275, AU148477, AA524480, AA476556, AW027610, BE207925,AW166935, AL521960, AI934516, AU149354, AW291597, AW974311, AA443023,AJ186348, AI597811, AI692241, N32579, BE619316, AI689448, AW152379,AI271524, AI093466, AI870536, AI149215, AI432467, AA854903, AA781886,AI199164, AI744310, AL043754, AI372057, BF725035, AI269272, AW770362,AA399350, AI367106, AA463464, AA512239, AW516985, W48832, AWL51757,AI086901, AW571473, AW514611, AA292378, AI079461, BE206384, AA934644,AI269712, AW189899, AA588341, AA676478, AAI26131, AW055258, AW166860,N70058, AA916656, AI874191, AW297040, AW083905, AW664480, AA427894,AA453450, AA843278, AI687474, AA620899, W49813, AA860641, AI692799,BE676833, AA306455, AA292016, T63718, AW236228, AW304861, AW772846,AW468035, BE964404, AI015326, AW662269, AA226903, AW090569, AA304032,AA915898, R56660, AW194056, AA293296, AA047874, H28877, AW076091,AI126745, AA482627, R50813, AA482480, AI674616, BF926901, AA045575,R68388, W37626, H01650, AA598596, AW364253, N59135, AL045883, N23722,AA908894, Z40636, R38774, R68593, AV708320, AI474604, R47766, AI611825,AA022933, BE829256, BF244825, AA888168, F03832, C04718, AA534374,BE297735, R50403, R56826, R81871, T33719, AI569605, N41917, AW731635,AW050454, BF737093, Z44868, AI648412, T06476, AA428005, W37625,BF893080, BF820895, BF769768, F07587, F01530, D54185, AI611777,AA902145, AA338785, Z25279, BE617105, F00464, AA084935, BE241564,R10512, AI627173, BE252263, AA022983, AV743083, BE940174, N75173,BE929227, 1128878, AA094149, AW294418, AI261764, AA449769, T63873,AI885556, AA775981, BE710635, BE936751, BC008836.1, BC001323.1,AL117480.1, AL132825.35, AF055022.1, AL096738.1, HMCAZ04 189 6682491-1287 15-1301 AL523496, AI804917, AW973481, AV716073, AW024415,AI302184, AV699357, AV700904, AW273859, AL523497, AA948547, BG236229,AW043839, AW516856, AI299954, BF108769, BE819542, AA911904, AA639665,AI133370, BE819457, BE819514, BE839222, AI097457, AI222830, BE819567,AW182738, AI522298, BE819505, AW119093, BF691126, AI305813, AW955102,W96028, BE839045, BE819511, BE831532, BF371410, N50841, BE819590,BE819554, AA527252, AA526991, AA643646, AI751876, AI290542, W94661,BE674172, AI240325, AI290551, AW050812, BF219419, T74688, AV659324,AI300130, AA885922, AAI00857, BE819468, BE772570, AA838720, AI400510,AA664347, C75117, AA056968, N54246, BE819545, AA481324, AA281439,AA486172, AI880359, AA468902, BE939493, AV660065, AV719793, T95295,D20113, BF379099, AA643688, AI394339, BE819461, BEB31675, AW854105,AW176080, T95375, AI890004, N58703, R50203, AW071048, AA280758, T74801,AW054934, T64005, Z19305, BE716219, AI635828, BE301581, AI928438,AA602300, AI685551, AW970142, AA514233, AV716521, BE819546, BG120804,BF027170, AW970145, BF752093, T36144, AI262366, BE789575, AV692708,R40496, BF912723, T86288, AW273588, AI273020, W45353, AA486109, T86387,T90950, T85836, AI635443, BF359777, AA587303, BE839081, AA533897,AW768388, T98225, R10150, T64084, BG169469, BE264000, BE819498,AA513774, AW945448, AA669130, AI872115, BE386262, T27349, BE819456,AA385681, AA253283, AI367142, AW316697, AI758444, W15319, AW467705,AV685386, AI819151, AI571469, BF359773, AV688204, BE546468, BG170954,AAI30233, AI557206, BE831556, BE831640, BE790051, AI613022, AA359423,BE008461, AI401483, AA576059, D82703, BF678342, AA353447, BF000110,BF359762, AA359712, BE018093, AV645442, AV645491, AA703984, AF151802.1,AF042284.1, AC090527.3, AC068722.6, HE8FD92 190 901142 1-3963 15-3977BG252727, BG260973, BG180624, BG122157, BE887548, AW948984, AW950907,BF965913, AI905469, BE869433, B0032175, AV718334, AV650921, BF793347,BE148077, BF793887, BG120463, BF204516, AW969050, BG121940, AL045343,AV649638, BE621758, AL037725, BE620934, BG169462, AV708063, AL045344,BE892224, AV708871, AV649666, AV653240, AV721974, BF740216, BG110357,AI888249, AV649944, AI814624, BE615208, BE613500, BE540375, AL043485,AA401244, BGL17844, AV705742, AV762287, AW614902, AI768623, AA404260,W95853, BE614234, AW167131, BE542745, BF844095, AW955266, BE551076,AI800419, AL040955, AL040932, BF891913, N32025, BF028570, BF980034,BE147946, AA315005, AL531145, AW020997, AI523819, BE162196, AV689195,AW196856, AL048907, BF378885, BE615086, AW851278, AW948982, AW069571,BF219231, AW664296, BF839881, BE299495, AI859769, AI804043, BE615260,AW439581, AI979070, AW873118, BG028790, AW173627, AW138481, BF836425,BE090183, AW470149, W95142, AI954566, AW966417, AW382691, AA431381,AI923608, AI920788, BF836432, BG028439, AI1074015, AW401596, AI096566,AA777093, AW168867, BE350092, AI872178, AL040146, BF832429, BG179393,AW196804, AJ634706, AI400065, AI983052, AL044108, BF571648, AW874528,AI200933, W94444, AI080172, AI073801, AV728394, BG107927, AA614709,AW963612, AAI34828, BE349345, AA463288, AA625480, AA953123, BE537489,AI589346, AW261884, BF757706, AI026817, AI654244, BE140097, AA932332,AV649957, AW002764, AI435532, N51228, AA725887, AI685954, AI247070,R97631, AI142752, AI569997, W81307, AI473795, AA962014, AA946853,AI916323, AW382688, AW954051, BF725969, AI000904, N69326, AA774786,AI445189, AI348365, BF433144, C17016, BE764894, AW193468, AI274717,AI420632, AI471592, AL040335, N94040, AAI34827, BE156276, W94957,BE151110, AW452484, AV726157, AI077452, AI675925, AW675613, AV694497,AV683792, AA868559, AA504467, AV684722, AI241864, AI826322, AA448916,AA872701, AA398843, BE966521, BF940653, BF804717, BG036364, AI865471,AW474946, AI088270, BE046420, BE764877, W45368, H27416, AV649926,AW020365, AV649742, AW241206, AV649875, BE857361, AA431829, BF923515,AI359458, AI439074, W94259, AL045559, BF998123, T78688, AI174662,AW377563, AV649995, BE764899, AI094039, AW797490, AA463197, AA494404,AI520933, AA609501, AV659915, AV662147, AI125505, BG111536, AI167605,AI149616, AV698723, AA609104, AI913467, BE835924, BF197860, AV651144,AA431425, AA975219, AA635251, BE710504, BE816681, D61350, AI811787,AV734255, AA341676, AV662120, AI368857, AA630621, AF131738.1,AB051480.1, AL117237.1, AL136890.1, AL022240.8, AL359752.11,AL049715.25, AB033071.1, AL050141.1, AK000726.1, Y14436.1, T49441,T49442, T65179, T90631, T79315, T79742, T83158, T85864, R16090, R15724,R18665, R22674, R38982, R39314, R41620, R43379, R41620, R43379, H24497,H29599, H44475, H65354, H65563, N69549, W17223, W40134, W92875, W95598,W95599, AA045010, AAI94855, AA470492, AA470959, AA483167, AA514556,AA557980, AA729817, AA738229, AA805073, AA908302, AA911490, AA938675,AA962188, AA976144, N55792, N74638, N83414, C01293, N88703, C14561,C14674, C15258, C15727, C15842, AA091964, AA094573, AA095891, AA247583,AA449426, AA599645, AA663625, AA665721, AA813724, AI003102, T10450,D31148, F11837, AA694588, AI251461, AI270073, AI280943, AI582507,AI126993, AI203813, AI247468.Description of Table 4

Table 4 provides a key to the tissue/cell source identifier codedisclosed in Table 1B.2, column 5. Column 1 provides the tissue/cellsource identifier code disclosed in Table 1B.2, Column 5. Columns 2-5provide a description of the tissue or cell source. Note that“Description” and “Tissue” sources (i.e. columns 2 and 3) having theprefix “a” indicates organs, tissues, or cells derived from “adult”sources. Codes corresponding to diseased tissues are indicated in column6 with the word “disease.” The use of the word “disease” in column 6 isnon-limiting. The tissue or cell source may be specific (e.g. aneoplasm), or may be disease-associated (e.g., a tissue sample from anormal portion of a diseased organ). Furthermore, tissues and/or cellslacking the “disease” designation may still be derived from sourcesdirectly or indirectly involved in a disease state or disorder, andtherefore may have a further utility in that disease state or disorder.In numerous cases where the tissue/cell source is a library, column 7identifies the vector used to generate the library. TABLE 4 CodeDescription Tissue Organ Cell Line Disease Vector AR022 a_Heart a_HeartAR023 a_Liver a_Liver AR024 a_mammary gland a_mammary gland AR025a_Prostate a_Prostate AR026 a_small intestine a_small intestine AR027a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood Bcells activated Blood B cells activated AR030 Blood B cells restingBlood B cells resting AR031 Blood T cells activated Blood T cellsactivated AR032 Blood T cells resting Blood T cells resting AR033 brainbrain AR034 breast breast AR035 breast cancer breast cancer AR036 CellLine CAOV3 Cell Line CAOV3 AR037 cell line PA-1 cell line PA-1 AR038cell line transformed cell line transformed AR039 colon colon AR040colon (9808co65R) colon (9808co65R) AR041 colon (9809co15) colon(9809co15) AR042 colon cancer colon cancer AR043 colon cancer coloncancer (9808co64R) (9808co64R) AR044 colon cancer 9809co14 colon cancer9809co14 AR050 Donor II B Cells 24 hrs Donor II B Cells 24 hrs AR051Donor II B Cells 72 hrs Donor II B Cells 72 hrs AR052 Donor II B-Cells24 hrs. Donor II B-Cells 24 hrs. AR053 Donor II B-Cells 72 hrs Donor IIB-Cells 72 hrs AR054 Donor II Resting B Donor II Resting B Cells CellsAR055 Heart Heart AR056 Human Lung Human Lung (clonetech) (clonetech)AR057 Human Mammary Human Mammary (clontech) (clontech) AR058 HumanThymus Human Thymus (clonetech) (clonetech) AR059 Jurkat (unstimulated)Jurkat (unstimulated) AR060 Kidney Kidney AR061 Liver Liver AR062 Liver(Clontech) Liver (Clontech) AR063 Lymphocytes chronic Lymphocyteschronic lymphocytic leukaemia lymphocytic leukaemia AR064 Lymphocytesdiffuse Lymphocytes diffuse large large B cell lymphoma B cell lymphomaAR065 Lymphocytes follicular Lymphocytes follicular lymphoma lymphomaAR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian(4004901) (4004901) AR068 Normal Ovary Normal Ovary 9508G045 9508G045AR069 Normal Ovary Normal Ovary 9701G208 9701G208 AR070 Normal OvaryNormal Ovary 9806G005 9806G005 AR071 Ovarian Cancer Ovarian Cancer AR072Ovarian Cancer Ovarian Cancer (9702G001) (9702G001) AR073 Ovarian CancerOvarian Cancer (9707G029) (9707G029) AR074 Ovarian Cancer Ovarian Cancer

AR075 Ovarian Cancer Ovarian Cancer (9806G019) (9806G019) AR076 OvarianCancer Ovarian Cancer (9807G017) (9807G017) AR077 Ovarian Cancer OvarianCancer (9809G001) (9809G001) AR078 ovarian cancer 15799 ovarian cancer15799 AR079 Ovarian Cancer Ovarian Cancer 17717AID 17717AID AR080Ovarian Cancer Ovarian Cancer 4004664B1 4004664B1 AR081 Ovarian CancerOvarian Cancer 4005315A1 4005315A1 AR082 ovarian cancer ovarian cancer94127303 94127303 AR083 Ovarian Cancer Ovarian Cancer 96069304 96069304AR084 Ovarian Cancer Ovarian Cancer 9707G029 9707G029 AR085 OvarianCancer Ovarian Cancer 9807G045 9807G045 AR086 ovarian cancer ovariancancer 9809G001 9809G001 AR087 Ovarian Cancer Ovarian Cancer 9905C032RC9905C032RC AR088 Ovarian cancer 9907 Ovarian cancer 9907 C00 C00 3rd 3rdAR089 Prostate Prostate AR090 Prostate (clonetech) Prostate (clonetech)AR091 prostate cancer prostate cancer AR092 prostate cancer #15176prostate cancer #15176 AR093 prostate cancer #15509 prostate cancer#15509

AR095 Small Intestine Small Intestine (Clontech) (Clontech) AR096 SpleenSpleen AR097 Thymus T cells Thymus T cells activated activated AR098Thymus T cells resting Thymus T cells resting AR099 Tonsil Tonsil AR100Tonsil geminal center Tonsil geminal center centroblast centroblastAR101 Tonsil germinal center Tonsil germinal center B B cell cell AR102Tonsil lymph node Tonsil lymph node AR103 Tonsil memory B cell Tonsilmemory B cell AR104 Whole Brain Whole Brain AR105 Xenograft ES-2Xenograft ES-2 AR106 Xenograft SW626 Xenograft SW626 AR119 001: IL-2001: IL-2 AR120 001: IL-2.1 001: IL-2.1 AR121 001: IL-2_b 001: IL-2_bAR124 002: Monocytes 002: Monocytes untreated untreated (1 hr) (1 hr)AR125 002: Monocytes 002: Monocytes untreated untreated (5 hrs) (5 hrs)AR126 002: Control.1C 002: Control.1C AR127 002: IL2.1C 002: IL2.1CAR130 003: Placebo-treated 003: Placebo-treated Rat Rat Lacrimal GlandLacrimal Gland AR131 003: Placebo-treated 003: Placebo-treated Rat RatSubmandibular Submandibular Gland Gland AR135 004: Monocytes 004:Monocytes untreated untreated (5 hrs) (5 hrs)

untreated 1 hr 1 hr AR139 005: Placebo (48 hrs) 005: Placebo (48 hrs)AR140 006: pC4 (24 hrs) 006: pC4 (24 hrs) AR141 006: pC4 (48 hrs) 006:pC4 (48 hrs) AR152 007: PHA(1 hr) 007: PHA(1 hr) AR153 007: PHA(6 HRS)007: PHA(6 HRS) AR154 007: PMA(6 hrs) 007: PMA(6 hrs) AR155 008: 1449_#2008: 1449_#2 AR161 01: A - max 24 01: A - max 24 AR162 01: A - max 2601: A - max 26 AR163 01: A - max 30 01: A - max 30 AR164 01: B - max 2401: B - max 24 AR165 01: B - max 26 01: B - max 26 AR166 01: B - max 3001: B - max 30 AR167 1449 Sample 1449 Sample AR168 3T3P10 1.0 uM insulin3T3P10 1.0 uM insulin AR169 3T3P10 10 nM Insulin 3T3P10 10 nM InsulinAR170 3T3P10 10 uM insulin 3T3P10 10 uM insulin AR171 3T3P10 No Insulin3T3P10 No Insulin AR172 3T3P4 3T3P4 AR173 Adipose (41892) Adipose(41892) AR174 Adipose Diabetic Adipose Diabetic (41611) (41611) AR175Adipose Diabetic Adipose Diabetic (41661) (41661) AR176 Adipose DiabeticAdipose Diabetic (41689) (41689) AR177 Adipose Diabetic Adipose Diabetic(41706) (41706) AR178 Adipose Diabetic Adipose Diabetic (42352) (42352)

(42366) AR180 Adipose Diabetic Adipose Diabetic (42452) (42452) AR181Adipose Diabetic Adipose Diabetic (42491) (42491) AR182 Adipose NormalAdipose Normal (41843) (41843) AR183 Adipose Normal Adipose Normal(41893) (41893) AR184 Adipose Normal Adipose Normal (42452) (42452)AR185 Adrenal Gland Adrenal Gland AR186 Adrenal Gland + Whole AdrenalGland + Whole Brain Brain AR187 B7(1 hr)+ (inverted) B7(1 hr)+(inverted) AR188 Breast (18275A2B) Breast (18275A2B) AR189 Breast(4004199) Breast (4004199) AR190 Breast (4004399) Breast (4004399) AR191Breast (4004943B7) Breast (4004943B7) AR192 Breast (4005570B1) Breast(4005570B1) AR193 Breast Cancer Breast Cancer (4004127A30) (4004127A30)AR194 Breast Cancer Breast Cancer (400443A21) (400443A21) AR195 BreastCancer Breast Cancer (4004643A2) (4004643A2) AR196 Breast Cancer BreastCancer (4004710A7) (4004710A7) AR197 Breast Cancer Breast Cancer(4004943A21) (4004943A21) AR198 Breast Cancer Breast Cancer (400553A2)(400553A2)

(9805C046R) (9805C046R) AR200 Breast Cancer Breast Cancer (9806C012R)(9806C012R) AR201 Breast Cancer (ODQ Breast Cancer (ODQ 45913) 45913)AR202 Breast Cancer Breast Cancer (ODQ45913) (ODQ45913) AR203 BreastCancer Breast Cancer (ODQ4591B) (ODQ4591B) AR204 Colon Cancer (15663)Colon Cancer (15663) AR205 Colon Cancer Colon Cancer (4005144A4)(4005144A4) AR206 Colon Cancer Colon Cancer (4005413A4) (4005413A4)AR207 Colon Cancer Colon Cancer (4005570B1) (4005570B1) AR208 ControlRNA #1 Control RNA #1 AR209 Control RNA #2 Control RNA #2 AR210 CulturedPreadipocyte Cultured Preadipocyte (blue) (blue) AR211 CulturedPreadipocyte Cultured Preadipocyte (Red) (Red) AR212 Donor II B-Cells 24hrs Donor II B-Cells 24 hrs AR213 Donor II Resting B- Donor II RestingB-Cells Cells AR214 H114EP12 10 nM H114EP12 10 nM Insulin Insulin AR215H114EP12 (10 nM H114EP12 (10 nM insulin) insulin) AR216 H114EP12 (2.6ug/ul) H114EP12 (2.6 ug/ul) AR217 H114EP12 (3.6 ug/ul) H114EP12 (3.6ug/ul) AR218 HUVEC #1 HUVEC #1

AR221 L6 undiff. L6 undiff. AR222 L6 Undifferentiated L6Undifferentiated AR223 L6P8 + 10 nM Insulin L6P8 + 10 nM Insulin AR224L6P8 + HS L6P8 + HS AR225 L6P8 10 nM Insulin L6P8 10 nM Insulin AR226Liver (00-06-A007B) Liver (00-06-A007B) AR227 Liver (96-02-A075) Liver(96-02-A075) AR228 Liver (96-03-A144) Liver (96-03-A144) AR229 Liver(96-04-A138) Liver (96-04-A138) AR230 Liver (97-10-A074B) Liver(97-10-A074B) AR231 Liver (98-09-A242A) Liver (98-09-A242A) AR232 LiverDiabetic (1042) Liver Diabetic (1042) AR233 Liver Diabetic (41616) LiverDiabetic (41616) AR234 Liver Diabetic (41955) Liver Diabetic (41955)AR235 Liver Diabetic (42352R) Liver Diabetic (42352R) AR236 LiverDiabetic (42366) Liver Diabetic (42366) AR237 Liver Diabetic (42483)Liver Diabetic (42483) AR238 Liver Diabetic (42491) Liver Diabetic(42491) AR239 Liver Diabetic (99-09- Liver Diabetic (99-09- A281A)A281A) AR240 Lung Lung AR241 Lung (27270) Lung (27270) AR242 Lung(2727Q) Lung (2727Q) AR243 Lung Cancer Lung Cancer (4005116A1)(4005116A1) AR244 Lung Cancer Lung Cancer (4005121A5) (4005121A5) AR245Lung Cancer Lung Cancer (4005121A5)) (4005121A5)) AR246 Lung Cancer LungCancer (4005340A4) (4005340A4)

AR248 Monocyte (CT) Monocyte (CT) AR249 Monocyte (OCT) Monocyte (OCT)AR250 Monocytes (CT) Monocytes (CT) AR251 Monocytes (INFG 18 hr)Monocytes (INFG 18 hr) AR252 Monocytes (INFG 18 hr) Monocytes (INFG 18hr) AR253 Monocytes (INFG 8-11) Monocytes (INFG 8-11) AR254 Monocytes (OCT) Monocytes (O CT) AR255 Muscle (91-01-A105) Muscle (91-01-A105) AR256Muscle (92-04-A059) Muscle (92-04-A059) AR257 Muscle (97-11-A056d)Muscle (97-11-A056d) AR258 Muscle (99-06-A210A) Muscle (99-06-A210A)AR259 Muscle (99-07-A203B) Muscle (99-07-A203B) AR260 Muscle(99-7-A203B) Muscle (99-7-A203B) AR261 Muscle Diabetic Muscle Diabetic(42352R) (42352R) AR262 Muscle Diabetic Muscle Diabetic (42366) (42366)AR263 NK-19 Control NK-19 Control AR264 NK-19 IL Treated 72 hrs NK-19 ILTreated 72 hrs AR265 NK-19 UK Treated 72 hrs. NK-19 UK Treated 72 hrs.AR266 Omentum Normal (94- Omentum Normal (94-08- 08-B009) B009) AR267Omentum Normal (97- Omentum Normal (97-01- 01-A039A) A039A) AR268Omentum Normal (97- Omentum Normal (97-04- 04-A114C) A114C) AR269Omentum Normal (97- Omentum Normal (97-06- 06-A117C) A117C) AR270Omentum Normal (97- Omentum Normal (97-09- 09-B004C) B004C)

(17717AID) (17717AID) AR272 Ovarian Cancer Ovarian Cancer (9905C023RC)(9905C023RC) AR273 Ovarian Cancer Ovarian Cancer (9905C032RC)(9905C032RC) AR274 Ovary (9508G045) Ovary (9508G045) AR275 Ovary(9701G208) Ovary (9701G208) AR276 Ovary 9806G005 Ovary 9806G005 AR277Pancreas Pancreas AR278 Placebo Placebo AR279 rIL2 Control rIL2 ControlAR280 RSS288L RSS288L AR281 RSS288LC RSS288LC AR282 Salivary GlandSalivary Gland AR283 Skeletal Muscle Skeletal Muscle AR284 SkeletalMuscle (91- Skeletal Muscle (91-01- 01-A105) A105) AR285 Skeletal Muscle(42180) Skeletal Muscle (42180) AR286 Skeletal Muscle (42386) SkeletalMuscle (42386) AR287 Skeletal Muscle (42461) Skeletal Muscle (42461)AR288 Skeletal Muscle (91-01- Skeletal Muscle (91-01- A105) A105) AR289Skeletal Muscle (92-04- Skeletal Muscle (92-04- A059) A059) AR290Skeletal Muscle (96-08- Skeletal Muscle (96-08- A171) A171) AR291Skeletal Muscle (97-07- Skeletal Muscle (97-07- A190A) A190A) AR292Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42352) (42352) AR293Skeletal Muscle Skeletal Muscle Diabetic Diabetic (42366) (42366)

Diabetic (42395) (42395) AR295 Skeletal Muscle Skeletal Muscle DiabeticDiabetic (42483) (42483) AR296 Skeletal Muscle Skeletal Muscle DiabeticDiabetic (42491) (42491) AR297 Skeletal Muscle Skeletal Muscle DiabeticDiabetic 42352 42352 AR298 Skeletal Musle (42461) Skeletal Musle (42461)AR299 Small Intestine Small Intestine AR300 Stomach Stomach AR301T-Cell + HDPBQ71.fc T-Cell + HDPBQ71.fc 1449 16 hrs 1449 16 hrs AR302T-Cell + HDPBQ71.fc T-Cell + HDPBQ71.fc 1449 6 hrs 1449 6 hrs AR303T-Cell + IL2 16 hrs T-Cell + IL2 16 hrs AR304 T-Cell + IL2 6 hrsT-Cell + IL2 6 hrs AR306 T-Cell Untreated 16 hrs T-Cell Untreated 16 hrsAR307 T-Cell Untreated 6 hrs T-Cell Untreated 6 hrs AR308 T-Cells 24hours T-Cells 24 hours AR309 T-Cells 24 hrs T-Cells 24 hrs AR310 T-Cells24 hrs. T-Cells 24 hrs. AR311 T-Cells 24 hrs T-Cells 24 hrs AR312T-Cells 4 days T-Cells 4 days AR313 Thymus Thymus AR314 TRE TRE AR315TREC TREC AR316 Virtual Mixture Virtual Mixture AR317 B lymphocyte, Blymphocyte, AR318 (non-T; non-B) (non-T; non-B) AR326 001-293 RNA(Vector 001-293 RNA (Vector Control) Control)

AR328 001: Control.1 001: Control.1 AR355 Acute Lymphocyte AcuteLymphocyte Leukemia Leukemia AR356 AML Patient #11 AML Patient #11 AR357AML Patient #2 AML Patient #2 AR358 AML Patient #2 SGAH AML Patient #2SGAH AR359 AML Patient#2 AML Patient#2 AR360 Aorta Aorta AR361 B Cell BCell AR362 B lymphoblast B lymphoblast AR363 B lymphocyte B lymphocyteAR364 B lymphocytes B lymphocytes AR365 B-cell B-cell AR366 B-CellsB-Cells AR367 B-Lymphoblast B-Lymphoblast AR368 B-LymphocytesB-Lymphocytes AR369 Bladder Bladder AR370 Bone Marrow Bone Marrow AR371Bronchial Epithelial Bronchial Epithelial Cell Cell AR372 BronchialEpithelial Bronchial Epithelial Cells Cells AR373 Caco-2A Caco-2A AR374Caco-2B Caco-2B AR375 Caco-2C Caco-2C AR376 Cardiac #1 Cardiac #1 AR377Cardiac #2 Cardiac #2 AR378 Chest Muscle Chest Muscle AR381 DendriticCell Dendritic Cell AR382 Dendritic cells Dendritic cells AR383 E. coliE. coli AR384 Epithelial Cells Epithelial Cells AR385 EsophagusEsophagus AR386 FPPS FPPS AR387 FPPSC FPPSC AR388 HepG2 Cell Line HepG2Cell Line AR389 HepG2 Cell line Buffer HepG2 Cell line Buffer 1 hr. 1hr. AR390 HepG2 Cell line Buffer HepG2 Cell line Buffer 06 hr. 06 hrAR391 HepG2 Cell line Buffer HepG2 Cell line Buffer 24 hr. 24 hr. AR392HepG2 Cell line Insulin HepG2 Cell line Insulin 01 hr. 01 hr. AR393HepG2 Cell line Insulin HepG2 Cell line Insulin 06 hr. 06 hr. AR394HepG2 Cell line Insulin HepG2 Cell line Insulin 24 hr. 24 hr. AR398HMC-1 HMC-1 AR399 HMCS HMCS AR400 HMSC HMSC AR401 HUVEC #3 HUVEC #3AR402 HUVEC #4 HUVEC #4 AR404 KIDNEY NORMAL KIDNEY NORMAL AR405 KIDNEYTUMOR KIDNEY TUMOR AR406 KIDNEY TUMOR AR407 Lymph Node Lymph Node AR408Macrophage Macrophage AR409 Megakarioblast Megakarioblast AR410 MonocyteMonocyte AR411 Monocytes Monocytes

AR413 Myocardium #3 Myocardium #3 AR414 Myocardium #4 Myocardium #4AR415 Myocardium #5 Myocardium #5 AR416 NK NK AR417 NK cell NK cellAR418 NK cells NK cells AR419 NKYa NKYa AR420 NKYa019 NKYa019 AR421Ovary Ovary AR422 Patient #11 Patient #11 AR423 Peripheral bloodPeripheral blood AR424 Primary Adipocytes Primary Adipocytes AR425Promyeloblast Promyeloblast AR427 RSSWT RSSWT AR428 RSSWTC RSSWTC AR429SW 480(G1) SW 480(G1) AR430 SW 480(G2) SW 480(G2) AR431 SW 480(G3) SW480(G3) AR432 SW 480(G4) SW 480(G4) AR433 SW 480(G5) SW 480(G5) AR434 TLymphoblast T Lymphoblast AR435 T Lymphocyte T Lymphocyte AR436 T-CellT-Cell AR438 T-Cell, T-Cell, AR439 T-Cells T-Cells AR440 T-lymphoblastT-lymphoblast AR441 Th 1 Th 1 AR442 Th 2 Th 2 AR443 Th1 Th1 AR444 Th2Th2 H0002 Human Adult Heart Human Adult Heart Heart Uni-ZAP XR

H0004 Human Adult Spleen Human Adult Spleen Spleen Uni-ZAP XR H0008Whole 6 Week Old Uni-ZAP XR Embryo H0009 Human Fetal Brain Uni-ZAP XRH0012 Human Fetal Kidney Human Fetal Kidney Kidney Uni-ZAP XR H0013Human 8 Week Whole Human 8 Week Old Embryo Uni-ZAP XR Embryo EmbryoH0014 Human Gall Bladder Human Gall Bladder Gall Bladder Uni-ZAP XRH0015 Human Gall Bladder, Human Gall Bladder Gall Bladder Uni-ZAP XRfraction II H0018 Human Greater Human Greater Omentum peritoneum Uni-ZAPXR Omentum, fII remake H0019 Human Fetal Heart Human Fetal Heart HeartpBluescript H0022 Jurkat Cells Jurkat T-Cell Line Lambda ZAP II H0024Human Fetal Lung III Human Fetal Lung Lung Uni-ZAP XR H0026 NamalwaCells Namalwa B-Cell Line, Lambda ZAP II EBV immortalized H0028 HumanOld Ovary Human Old Ovary Ovary pBluescript H0030 Human Placenta Uni-ZAPXR H0031 Human Placenta Human Placenta Placenta Uni-ZAP XR H0032 HumanProstate Human Prostate Prostate Uni-ZAP XR H0033 Human Pituitary HumanPituitary Uni-ZAP XR H0036 Human Adult Small Human Adult Small SmallInt. Uni-ZAP XR Intestine Intestine H0038 Human Testes Human TestesTestis Uni-ZAP XR H0039 Human Pancreas Tumor Human Pancreas TumorPancreas disease Uni-ZAP XR H0040 Human Testes Tumor Human Testes TumorTestis disease Uni-ZAP XR H0041 Human Fetal Bone Human Fetal Bone BoneUni-ZAP XR H0042 Human Adult Human Adult Pulmonary Lung Uni-ZAP XRPulmonary H0046 Human Endometrial Human Endometrial Tumor Uterus diseaseUni-ZAP XR

H0050 Human Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0051 HumanHippocampus Human Hippocampus Brain Uni-ZAP XR H0052 Human CerebellumHuman Cerebellum Brain Uni-ZAP XR H0056 Human Umbilical Vein, HumanUmbilical Vein Umbilical vein Uni-ZAP XR Endo. remake Endothelial CellsH0057 Human Fetal Spleen Uni-ZAP XR H0059 Human Uterine Cancer HumanUterine Cancer Uterus disease Lambda ZAP II H0060 Human Macrophage HumanMacrophage Blood Cell Line pBluescript H0061 Human Macrophage HumanMacrophage Blood Cell Line pBluescript H0063 Human Thymus Human ThymusThymus Uni-ZAP XR H0064 Human Right Human Brain, right Brain Uni-ZAP XRHemisphere of Brain hemisphere H0068 Human Skin Tumor Human Skin TumorSkin disease Uni-ZAP XR H0069 Human Activated T- Activated T-Cells BloodCell Line Uni-ZAP XR Cells H0071 Human Infant Adrenal Human InfantAdrenal Adrenal gland Uni-ZAP XR Gland Gland H0075 Human Activated T-Activated T-Cells Blood Cell Line Uni-ZAP XR Cells (II) H0081 HumanFetal Epithelium Human Fetal Skin Skin Uni-ZAP XR (Skin) H0082 HumanFetal Muscle Human Fetal Muscle Sk Muscle Uni-ZAP XR H0083 HUMAN JURKATJurkat Cells Uni-ZAP XR MEMBRANE BOUND POLYSOMES H0085 Human Colon HumanColon Lambda ZAP II H0086 Human epithelioid Epithelioid Sarcoma, SkMuscle disease Uni-ZAP XR sarcoma muscle H0087 Human Thymus Human ThymuspBluescript H0090 Human T-Cell T-Cell Lymphoma T-Cell disease Uni-ZAP XRLymphoma H0095 Human Greater Human Greater Omentum peritoneum Uni-ZAP XR

Remake H0098 Human Adult Liver, Human Adult Liver Liver Uni-ZAP XRsubtracted H0100 Human Whole Six Human Whole Six Week Embryo Uni-ZAP XRWeek Old Embryo Old Embryo H0101 Human 7 Weeks Old Human Whole 7 WeekOld Embryo Lambda ZAP II Embryo, subtracted Embryo H0102 Human Whole 6Week Human Whole Six Week Embryo pBluescript Old Embryo (II), subt OldEmbryo H0105 Human Fetal Heart, Human Fetal Heart Heart pBluescriptsubtracted H0107 Human Infant Adrenal Human Infant Adrenal Adrenal glandpBluescript Gland, subtracted Gland H0117 Human Uterine Cancer, HumanUterine Cancer Uterus pBluescript subtracted H0121 Human Cornea, HumanCornea eye Uni-ZAP XR subtracted H0122 Human Adult Skeletal HumanSkeletal Muscle Sk Muscle Uni-ZAP XR Muscle H0123 Human Fetal Dura HumanFetal Dura Mater Brain Uni-ZAP XR Mater H0124 Human Human Sk Muscledisease Uni-ZAP XR Rhabdomyosarcoma Rhabdomyosarcoma H0125 Cem cellsCyclohexamide Treated Blood Cell Line Uni-ZAP XR cyclohexamide treatedCem, Jurkat, Raji, and Supt H0129 Jurkat cells, thiouridine Jurkat CellsUni-ZAP XR activated, fract II H0130 LNCAP untreated LNCAP Cell LineProstate Cell Line Uni-ZAP XR H0131 LNCAP + o.3 nM R1881 LNCAP Cell LineProstate Cell Line Uni-ZAP XR H0132 LNCAP + 30 nM R1881 LNCAP Cell LineProstate Cell Line Uni-ZAP XR H0134 Raji Cells, Cyclohexamide TreatedBlood Cell Line Uni-ZAP XR cyclohexamide treated Cem, Jurkat, Raji, andSupt

Sarcoma H0136 Supt Cells, Cyclohexamide Treated Blood Cell Line Uni-ZAPXR cyclohexamide treated Cem, Jurkat, Raji, and Supt H0140 ActivatedT-Cells, 8 hrs. Activated T-Cells Blood Cell Line Uni-ZAP XR H0141Activated T-Cells, 12 hrs. Activated T-Cells Blood Cell Line Uni-ZAP XRH0144 Nine Week Old Early 9 Wk Old Early Stage Embryo Uni-ZAP XR StageHuman Human H0147 Human Adult Liver Human Adult Liver Liver Uni-ZAP XRH0149 7 Week Old Early Stage Human Whole 7 Week Old Embryo Uni-ZAP XRHuman, subtracted Embryo H0150 Human Epididymus Epididymis TestisUni-ZAP XR H0151 Early Stage Human Human Fetal Liver Liver Uni-ZAP XRLiver H0154 Human Fibrosarcoma Human Skin Fibrosarcoma Skin diseaseUni-ZAP XR H0156 Human Adrenal Gland Human Adrenal Gland Adrenal Glanddisease Uni-ZAP XR Tumor Tumor H0159 Activated T-Cells, 8 hrs.,Activated T-Cells Blood Cell Line Uni-ZAP XR ligation 2 H0163 HumanSynovium Human Synovium Synovium Uni-ZAP XR H0165 Human Prostate Cancer,Human Prostate Cancer, Prostate disease Uni-ZAP XR Stage B2 stage B2H0166 Human Prostate Cancer, Human Prostate Cancer, Prostate diseaseUni-ZAP XR Stage B2 fraction stage B2 H0167 Activated T-Cells, 24 hrs.Activated T-Cells Blood Cell Line Uni-ZAP XR H0169 Human ProstateCancer, Human Prostate Cancer, Prostate disease Uni-ZAP XR Stage Cfraction stage C H0170 12 Week Old Early Twelve Week Old Early EmbryoUni-ZAP XR Stage Human Stage Human H0171 12 Week Old Early Twelve WeekOld Early Embryo Uni-ZAP XR Stage Human, II Stage Human

Cardiomyopathy, RNA remake H0177 CAMA1Ee Cell Line CAMA1Ee Cell LineBreast Cell Line Uni-ZAP XR H0178 Human Fetal Brain Human Fetal BrainBrain Uni-ZAP XR H0179 Human Neutrophil Human Neutrophil Blood Cell LineUni-ZAP XR H0181 Human Primary Breast Human Primary Breast Breastdisease Uni-ZAP XR Cancer Cancer H0182 Human Primary Breast HumanPrimary Breast Breast disease Uni-ZAP XR Cancer Cancer H0183 Human ColonCancer Human Colon Cancer Colon disease Uni-ZAP XR H0187 Resting T-CellT-Cells Blood Cell Line Lambda ZAP II H0188 Human Normal Breast HumanNormal Breast Breast Uni-ZAP XR H0194 Human Cerebellum, Human CerebellumBrain pBluescript subtracted H0196 Human Human Cardiomyopathy HeartUni-ZAP XR Cardiomyopathy, subtracted H0197 Human Fetal Liver, HumanFetal Liver Liver Uni-ZAP XR subtracted H0199 Human Fetal Liver, HumanFetal Liver Liver Uni-ZAP XR subtracted, neg clone H0200 Human GreaterHuman Greater Omentum peritoneum Uni-ZAP XR Omentum, fract II remake,H0201 Human Hippocampus, Human Hippocampus Brain pBluescript subtractedH0202 Jurkat Cells, Cyclohexamide Treated Blood Cell Line Uni-ZAP XRcyclohexamide treated, Cem, Jurkat, Raji, and Supt subtraction H0204Human Colon Cancer, Human Colon Cancer Colon pBluescript subtractedH0205 Human Colon Cancer, Human Colon Cancer Colon pBluescript

H0208 Early Stage Human Human Fetal Lung Lung pBluescript Lung,subtracted H0209 Human Cerebellum, Human Cerebellum Brain Uni-ZAP XRdifferentially expressed H0212 Human Prostate, Human Prostate ProstatepBluescript subtracted H0213 Human Pituitary, Human Pituitary Uni-ZAP XRsubtracted H0216 Supt cells, Cyclohexamide Treated Blood Cell LinepBluescript cyclohexamide treated, Cem, Jurkat, Raji, and Suptsubtracted H0220 Activated T-Cells, 4 hrs, Activated T-Cells Blood CellLine Uni-ZAP XR subtracted H0222 Activated T-Cells, 8 hrs, ActivatedT-Cells Blood Cell Line Uni-ZAP XR subtracted H0224 Activated T-Cells,12 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XR subtracted H0225Activated T-Cells, Activated T-Cells Blood Cell Line Uni-ZAP XR 12 hrs,differentially expressed H0229 Early Stage Human Early Stage Human BrainBrain Lambda ZAP II Brain, random primed H0230 Human HumanCardiomyopathy Heart disease Uni-ZAP XR Cardiomyopathy, diff exp H0231Human Colon, Human Colon pBluescript subtraction H0235 Human coloncancer, Human Colon Cancer, Liver pBluescript metaticized to liver,metasticized to liver subtraction H0239 Human Kidney Tumor Human KidneyTumor Kidney disease Uni-ZAP XR H0241 C7MCF7 cell line, C7MCF7 CellLine, Breast Cell Line Uni-ZAP XR

subtraction H0242 Human Fetal Heart, Human Fetal Heart Heart pBluescriptDifferential (Fetal- 0 Specific) H0244 Human 8 Week Whole Human 8 WeekOld Embryo Uni-ZAP XR Embryo, subtracted Embryo H0246 Human Fetal Liver-Human Fetal Liver Liver Uni-ZAP XR Enzyme subtraction H0249 HE7,subtracted by Human Whole 7 Week Old Embryo Uni-ZAP XR hybridizationwith E7 Embryo cDNA H0250 Human Activated Human Monocytes Uni-ZAP XRMonocytes H0251 Human Human Chondrosarcoma Cartilage disease Uni-ZAP XRChondrosarcoma H0252 Human Osteosarcoma Human Osteosarcoma Bone diseaseUni-ZAP XR H0253 Human adult testis, Human Adult Testis Testis Uni-ZAPXR large inserts H0254 Breast Lymph node Breast Lymph Node Lymph NodeUni-ZAP XR cDNA library H0255 breast lymph node Breast Lymph Node LymphNode Lambda ZAP II CDNA library H0256 HL-60, unstimulated Human HL-60Cells, Blood Cell Line Uni-ZAP XR unstimulated H0257 HL-60, PMA 4H HL-60Cells, PMA Blood Cell Line Uni-ZAP XR stimulated 4H H0261 H. cerebellum,Enzyme Human Cerebellum Brain Uni-ZAP XR subtracted H0263 human coloncancer Human Colon Cancer Colon disease Lambda ZAP II H0264 humantonsils Human Tonsil Tonsil Uni-ZAP XR H0265 Activated T-Cell T-CellsBlood Cell Line Uni-ZAP XR (12 hs)/Thiouridine

H0266 Human Microvascular HMEC Vein Cell Line Lambda ZAP II EndothelialCells, fract. A H0267 Human Microvascular HMEC Vein Cell Line Lambda ZAPII Endothelial Cells, fract. B H0268 Human Umbilical Vein HUVE CellsUmbilical vein Cell Line Lambda ZAP II Endothelial Cells, fract. A H0269Human Umbilical Vein HUVE Cells Umbilical vein Cell Line Lambda ZAP IIEndothelial Cells, fract. B H0270 HPAS (human pancreas, Human PancreasPancreas Uni-ZAP XR subtracted) H0271 Human Neutrophil, HumanNeutrophil - Blood Cell Line Uni-ZAP XR Activated Activated H0272 HUMANTONSILS, Human Tonsil Tonsil Uni-ZAP XR FRACTION 2 H0274 Human AdultSpleen, Human Adult Spleen Spleen Uni-ZAP XR fractionII H0275 HumanInfant Adrenal Human Infant Adrenal Adrenal gland pBluescript Gland,Subtracted Gland H0280 K562 + PMA (36 hrs) K562 Cell line cell line CellLine ZAP Express H0284 Human OB MG63 Human Osteoblastoma Bone Cell LineUni-ZAP XR control fraction I MG63 cell line H0286 Human OB MG63 HumanOsteoblastoma Bone Cell Line Uni-ZAP XR treated (10 nM E2) MG63 cellline fraction I H0288 Human OB HOS Human Osteoblastoma Bone Cell LineUni-ZAP XR control fraction I HOS cell line H0290 Human OB HOS treatedHuman Osteoblastoma Bone Cell Line Uni-ZAP XR (1 nM E2) fraction I HOScell line

(10 nM E2) fraction I HOS cell line H0293 WI 38 cells Uni-ZAP XR H0294Amniotic Cells - TNF Amniotic Cells - TNF Placenta Cell Line Uni-ZAP XRinduced induced H0295 Amniotic Cells - Amniotic Cells - Primary PlacentaCell Line Uni-ZAP XR Primary Culture Culture H0305 CD34 positive cellsCD34 Positive Cells Cord Blood ZAP Express (Cord Blood) H0306 CD34depleted Buffy CD34 Depleted Buffy Coat Cord Blood ZAP Express Coat(Cord Blood) (Cord Blood) H0309 Human Chronic Synovium, Chronic Synoviumdisease Uni-ZAP XR Synovitis Synovitis/Osteoarthritis H0316 HUMANSTOMACH Human Stomach Stomach Uni-ZAP XR H0318 HUMAN B CELL Human B CellLymphoma Lymph Node disease Uni-ZAP XR LYMPHOMA H0327 human corpuscolosum Human Corpus Callosum Brain Uni-ZAP XR H0328 human ovariancancer Ovarian Cancer Ovary disease Uni-ZAP XR H0329 DermatofibrosarcomaDermatofibrosarcoma Skin disease Uni-ZAP XR Protuberance ProtuberansH0331 Hepatocellular Tumor Hepatocellular Tumor Liver disease Lambda ZAPII H0333 Hemangiopericytoma Hemangiopericytoma Blood vessel diseaseLambda ZAP II H0334 Kidney cancer Kidney Cancer Kidney disease Uni-ZAPXR H0339 Duodenum Duodenum Uni-ZAP XR H0340 Corpus Callosum CorpusCollosum-93052 Uni-ZAP XR H0341 Bone Marrow Cell Line Bone Marrow CellLine Bone Marrow Cell Line Uni-ZAP XR (RS4;11) RS4;11 H0343 stomachcancer (human) Stomach Cancer - 5383A disease Uni-ZAP XR (human) H0344Adipose tissue (human) Adipose - 6825A (human) Uni-ZAP XR H0345 SKINSkin - 4000868H Skin Uni-ZAP XR H0346 Brain-medulloblastoma Brain(Medulloblastoma)- Brain disease Uni-ZAP XR 9405C006R

cDNA library H0350 Human Fetal Liver, Human Fetal Liver, mixed LiverUni-ZAP XR mixed 10 & 14 week 10&14 Week H0351 Glioblastoma GlioblastomaBrain disease Uni-ZAP XR H0352 wilm''s tumor Wilm''s Tumor diseaseUni-ZAP XR H0354 Human Leukocytes Human Leukocytes Blood Cell LinepCMVSport 1 H0355 Human Liver Human Liver, normal Adult pCMVSport 1H0357 H. Normalized Fetal Human Fetal Liver Liver Uni-ZAP XR Liver, IIH0361 Human rejected kidney Human Rejected Kidney disease pBluescriptH0366 L428 cell line L428 ZAP Express H0369 H. Atrophic AtrophicEndometrium and Uni-ZAP XR Endometrium myometrium H0370 H. Lymph nodebreast Lymph node with Met. disease Uni-ZAP XR Cancer Breast CancerH0373 Human Heart Human Adult Heart Heart pCMVSport 1 H0374 Human BrainHuman Brain pCMVSport 1 H0375 Human Lung Human Lung pCMVSport 1 H0379Human Tongue, frac 1 Human Tongue pSport1 H0380 Human Tongue, frac 2Human Tongue pSport1 H0381 Bone Cancer Bone Cancer disease Uni-ZAP XRH0383 Human Prostate BPH, Human Prostate BPH Uni-ZAP XR re-excisionH0385 H. Leukocytes, Kozak Human Leukocytes Blood Cell Line pCMVSport 1H0388 Human Rejected Human Rejected Kidney disease pBluescript Kidney,704 re-excision H0390 Human Amygdala Human Amygdala disease pBluescriptDepression, re-excision Depression H0391 H. Meniingima, M6 HumanMeningima brain pSport1 H0392 H. Meningima, M1 Human Meningima brainpSport1 H0393 Fetal Liver, subtraction Human Fetal Liver LiverpBluescript II

H0399 Human Kidney Cortex, Human Kidney Cortex Lambda ZAP II re-rescueH0400 Human Striatum Human Brain, Striatum Brain Lambda ZAP IIDepression, re-rescue Depression H0401 Human Pituitary, Human PituitarypBluescript subtracted V H0402 CD34 depleted Buffy CD34 Depleted BuffyCoat Cord Blood ZAP Express Coat (Cord Blood), re- (Cord Blood) excisionH0403 H. Umbilical Vein HUVE Cells Umbilical vein Cell Line Uni-ZAP XREndothelial Cells, IL4 induced H0406 H Amygdala Human Amygdala Uni-ZAPXR Depression, subtracted Depression H0408 Human kidney Cortex, HumanKidney Cortex pBluescript subtracted H0409 H. Striatum Depression, HumanBrain, Striatum Brain pBluescript subtracted Depression H0411 H FemaleBladder, Human Female Adult Bladder pSport1 Adult Bladder H0412 Humanumbilical vein HUVE Cells Umbilical vein Cell Line pSport1 endothelialcells, IL-4 induced H0413 Human Umbilical Vein HUVE Cells Umbilical veinCell Line pSport1 Endothelial Cells, uninduced H0414 Ovarian Tumor I,Ovarian Tumor, OV5232 Ovary disease pSport1 OV5232 H0415 H. OvarianTumor, II, Ovarian Tumor, OV5232 Ovary disease pCMVSport 2.0 OV5232H0416 Human Neutrophils, Human Neutrophil - Blood Cell Line pBluescriptActivated, re-excision Activated

subtracted VIII H0419 Bone Cancer, re- Bone Cancer Uni-ZAP XR excisionH0421 Human Bone Marrow, Bone Marrow pBluescript re-excision H0422T-Cell PHA 16 hrs T-Cells Blood Cell Line pSport1 H0423 T-Cell PHA 24hrs T-Cells Blood Cell Line pSport1 H0424 Human Pituitary, subt HumanPituitary pBluescript IX H0427 Human Adipose Human Adipose, left pSport1hiplipoma H0428 Human Ovary Human Ovary Tumor Ovary pSport1 H0431 H.Kidney Medulla, re- Kidney medulla Kidney pBluescript excision H0433Human Umbilical Vein HUVE Cells Umbilical vein Cell Line pBluescriptEndothelial cells, frac B, re-excision H0435 Ovarian Tumor 10-3-95Ovarian Tumor, OV350721 Ovary pCMVSport 2.0 H0436 Resting T-Cell T-CellsBlood Cell Line pSport1 Library, II H0437 H Umbilical Vein HUVE CellsUmbilical vein Cell Line Lambda ZAP II Endothelial Cells, frac A,re-excision H0438 H. Whole Brain #2, re- Human Whole Brain #2 ZAPExpress excision H0439 Human Eosinophils Eosinophils pBluescript H0441H. Kidney Cortex, Kidney cortex Kidney pBluescript subtracted H0444Spleen metastic Spleen, Metastic malignant Spleen disease pSport1melanoma melanoma H0445 Spleen, Chronic Human Spleen, CLL Spleen diseasepSport1 lymphocytic leukemia

H0450 CD34+cells, II CD34 positive cells pCMVSport 2.0 H0453 H. KidneyPyramid, Kidney pyramids Kidney pBluescript subtracted H0455 H. StriatumDepression, Human Brain, Striatum Brain pBluescript subt DepressionH0456 H Kidney Cortex, Human Kidney Cortex pBluescript subtracted IIIH0457 Human Eosinophils Human Eosinophils pSport1 H0458 CD34+ cell, I,frac II CD34 positive cells pSport1 H0459 CD34+ cells, II, CD34 positivecells pCMVSport 2.0 FRACTION 2 H0461 H. Kidney Medulla, Kidney medullaKidney pBluescript subtracted H0477 Human Tonsil, Lib 3 Human TonsilTonsil pSport1 H0478 Salivary Gland, Lib 2 Human Salivary Gland Salivarygland pSport1 H0483 Breast Cancer cell line, Breast Cancer Cell line,pSport1 MDA 36 MDA 36 H0484 Breast Cancer Cell line, Breast Cancer Cellline, pSport1 angiogenic Angiogenic, 36T3 H0485 Hodgkin''s Lymphoma IHodgkin''s Lymphoma I disease pCMVSport 2.0 H0486 Hodgkin''s LymphomaHodgkin''s Lymphoma II disease pCMVSport 2.0 II H0487 Human Tonsils, libI Human Tonsils pCMVSport 2.0 H0488 Human Tonsils, Lib 2 Human TonsilspCMVSport 2.0 H0492 HL-60, RA 4h, HL-60 Cells, RA stimulated Blood CellLine Uni-ZAP XR Subtracted for 4H H0494 Keratinocyte KeratinocytepCMVSport 2.0 H0497 HEL cell line HEL cell line HEL 92.1.7 pSport1 H0505Human Astrocyte Human Astrocyte pSport1 H0506 Ulcerative Colitis ColonColon pSport1 H0509 Liver, Hepatoma Human Liver, Hepatoma, Liver diseasepCMVSport 3.0 patient 8

Patient # 8 H0518 pBMC stimulated w/ pBMC stimulated with poly pCMVSport3.0 poly I/C I/C H0519 NTERA2, control NTERA2, Teratocarcinoma pCMVSport3.0 cell line H0520 NTERA2 + retinoic NTERA2, Teratocarcinoma pSport1acid, 14 days cell line H0521 Primary Dendritic Cells, Primary Dendriticcells pCMVSport 3.0 lib 1 H0522 Primary Dendritic Primary Dendriticcells pCMVSport 3.0 cells, frac 2 H0529 Myoloid Progenitor Cell TF-1Cell Line; Myoloid pCMVSport 3.0 Line progenitor cell line H0530 HumanDermal Human Dermal Endothelial pSport1 Endothelial Cells; untreatedCells, untreated H0535 Human ovary tumor cell Ovarian Tumor, OV350721Ovary disease pSport1 OV350721 H0538 Merkel Cells Merkel cells Lymphnode pSport1 H0539 Pancreas Islet Cell Pancreas Islet Cell TumourPancreas disease pSport1 Tumor H0540 Skin, burned Skin, leg burned SkinpSport1 H0542 T Cell helper I Helper T cell pCMVSport 3.0 H0543 T cellhelper II Helper T cell pCMVSport 3.0 H0544 Human endometrial Humanendometrial stromal pCMVSport 3.0 stromal cells cells H0545 Humanendometrial Human endometrial stromal pCMVSport 3.0 stromalcells-treated cells-treated with proge with progesterone H0546 Humanendometrial Human endometrial stromal pCMVSport 3.0 stromalcells-treated cells-treated with estra with estradiol

teratocarcinoma cell cell line line + retinoic acid (14 days) H0549 H.Epididiymus, caput Human Epididiymus, caput Uni-ZAP XR & corpus andcorpus H0550 H. Epididiymus, cauda Human Epididiymus, cauda Uni-ZAP XRH0551 Human Thymus Stromal Human Thymus Stromal pCMVSport 3.0 CellsCells H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 RejectedKidney, lib 4 Human Rejected Kidney Kidney disease pCMVSport 3.0 H0556Activated T- T-Cells Blood Cell Line Uni-ZAP XR cell(12 h)/Thiouridine-re-excision H0559 HL-60, PMA 4H, re- HL-60 Cells, PMA Blood Cell LineUni-ZAP XR excision stimulated 4H H0560 KMH2 KMH2 pCMVSport 3.0 H0561L428 L428 pCMVSport 3.0 H0562 Human Fetal Brain, Human Fetal BrainpCMVSport 2.0 normalized c5-11-26 H0563 Human Fetal Brain, Human FetalBrain pCMVSport 2.0 normalized 50021F H0565 HUman Fetal Brain, HumanFetal Brain pCMVSport 2.0 normalized 100024F H0566 Human Fetal HumanFetal Brain pCMVSport 2.0 Brain, normalized c50F H0567 Human FetalBrain, Human Fetal Brain pCMVSport 2.0 normalized A5002F H0569 HumanFetal Brain, Human Fetal Brain pCMVSport 2.0 normalized CO H0570 HumanFetal Brain, Human Fetal Brain pCMVSport 2.0 normalized C500H H0571Human Fetal Brain, Human Fetal Brain pCMVSport 2.0

H0572 Human Fetal Brain, Human Fetal Brain pCMVSport 2.0 normalizedAC5002 H0574 Hepatocellular Tumor; Hepatocellular Tumor Liver diseaseLambda ZAP II re-excision H0575 Human Adult Human Adult Pulmonary LungUni-ZAP XR Pulmonary; re-excision H0576 Resting T-Cell; re- T-CellsBlood Cell Line Lambda ZAP II excision H0580 Dendritic cells, pooledPooled dendritic cells pCMVSport 3.0 H0581 Human Bone Marrow, Human BoneMarrow Bone Marrow pCMVSport 3.0 treated H0583 B Cell lymphoma B CellLymphoma B Cell disease pCMVSport 3.0 H0584 Activated T-cells, 24 hrs,Activated T-Cells Blood Cell Line Uni-ZAP XR re-excision H0585 ActivatedT-Cells, 12 hrs, Activated T-Cells Blood Cell Line Uni-ZAP XRre-excision H0586 Healing groin wound, healing groin wound, 6.5 groindisease pCMVSport 3.0 6.5 hours post incision hours post incision - 2/H0587 Healing groin wound; Groin-2/19/97 groin disease pCMVSport 3.0 7.5hours post incision H0589 CD34 positive cells CD34 Positive Cells CordBlood ZAP Express (cord blood), re-ex H0590 Human adult small HumanAdult Small Small Int. Uni-ZAP XR intestine, re-excision Intestine H0591Human T-cell T-Cell Lymphoma T-Cell disease Uni-ZAP XR lymphoma;re-excision H0592 Healing groin wound - HGS wound healing diseasepCMVSport 3.0 zero hr post-incision project; abdomen (control) H0593Olfactory Olfactory epithelium from pCMVSport 3.0 epithelium;nasalcavity roof of left nasal cacit H0594 Human Lung Cancer; re- HumanLung Cancer Lung disease Lambda ZAP II

H0595 Stomach cancer Stomach Cancer - 5383A disease Uni-ZAP XR (human);re-excision (human) H0596 Human Colon Human Colon Cancer Colon LambdaZAP II Cancer; re-excision H0597 Human Colon; re- Human Colon Lambda ZAPII excision H0598 Human Stomach; re- Human Stomach Stomach Uni-ZAP XRexcision H0599 Human Adult Heart; re- Human Adult Heart Heart Uni-ZAP XRexcision H0600 Healing Abdomen Abdomen disease pCMVSport 3.0 wound;70&90 min post incision H0601 Healing Abdomen Abdomen disease pCMVSport3.0 Wound; 15 days post incision H0602 Healing Abdomen Abdomen diseasepCMVSport 3.0 Wound; 21&29 days post incision H0604 Human Pituitary, re-Human Pituitary pBluescript excision H0606 Human Primary Breast HumanPrimary Breast Breast disease Uni-ZAP XR Cancer; re-excision CancerH0607 H. Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 50A3 H0611H. Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 500 B H0613 H.Leukocytes, H. Leukocytes pCMVSport 1 normalized cot 5B H0615 HumanOvarian Cancer Ovarian Cancer Ovary disease Uni-ZAP XR Reexcision H0616Human Testes, Human Testes Testis Uni-ZAP XR

H0617 Human Primary Breast Human Primary Breast Breast disease Uni-ZAPXR Cancer Reexcision Cancer H0618 Human Adult Testes, Human Adult TestisTestis Uni-ZAP XR Large Inserts, Reexcision H0619 Fetal Heart HumanFetal Heart Heart Uni-ZAP XR H0620 Human Fetal Kidney; Human FetalKidney Kidney Uni-ZAP XR Reexcision H0622 Human Pancreas Human PancreasTumor Pancreas disease Uni-ZAP XR Tumor; Reexcision H0623 HumanUmbilical Vein; Human Umbilical Vein Umbilical vein Uni-ZAP XRReexcision Endothelial Cells H0624 12 Week Early Stage Twelve Week OldEarly Embryo Uni-ZAP XR Human II; Reexcision Stage Human H0625 Ku 812FBasophils Line Ku 812F Basophils pSport1 H0626 Saos2 Cells; UntreatedSaos2 Cell Line; Untreated pSport1 H0627 Saos2 Cells; Vitamin Saos2 CellLine; Vitamin pSport1 D3 Treated D3 Treated H0628 Human Pre- HumanPre-Differentiated Uni-ZAP XR Differentiated Adipocytes Adipocytes H0630Human Human Normalized pCMVSport 1 Leukocytes, normalized leukocytecontrol #4 H0631 Saos2, Dexamethosome Saos2 Cell Line; pSport1 TreatedDexamethosome Treated H0632 Hepatocellular Hepatocellular Tumor LiverLambda ZAP II Tumor; re-excision H0633 Lung Carcinoma A549 TNFalphaactivated A549-- disease pSport1 TNFalpha activated Lung Carcinoma H0634Human Testes Tumor, Human Testes Tumor Testis disease Uni-ZAP XRre-excision

Cells, re-excision H0637 Dendritic Cells From Dentritic cells from CD34pSport1 CD34 Cells cells H0638 CD40 activated CD40 activated monocytepSport1 monocyte dendridic dendridic cells cells H0640 Ficolled HumanStromal Ficolled Human Stromal Other Cells, Untreated Cells, UntreatedH0641 LPS activated derived LPS activated monocyte pSport1 dendriticcells derived dendritic cells H0642 Hep G2 Cells, lambda Hep G2 CellsOther library H0643 Hep G2 Cells, PCR Hep G2 Cells Other library H0644Human Placenta (re- Human Placenta Placenta Uni-ZAP XR excision) H0645Fetal Heart, re-excision Human Fetal Heart Heart Uni-ZAP XR H0646 Lung,Cancer (4005313 Metastatic squamous cell pSport1 A3): Invasive Poorlylung carcinoma, poorly di Differentiated Lung Adenocarcinoma, H0647Lung, Cancer (4005163 Invasive poorly disease pSport1 B7): Invasive,Poorly differentiated lung Diff. Adenocarcinoma, adenocarcinomaMetastatic H0648 Ovary, Cancer: Papillary Cstic neoplasm of diseasepSport1 (4004562 B6) Papillary low malignant potentia Serous CysticNeoplasm, Low Malignant Pot H0649 Lung, Normal: Normal Lung pSport1(4005313 B1)

H0651 Ovary, Normal: Normal Ovary pSport1 (9805C040R) H0652 Lung,Normal: Normal Lung pSport1 (4005313 B1) H0653 Stromal Cells StromalCells pSport1 H0656 B-cells (unstimulated) B-cells (unstimulated)pSport1 H0657 B-cells (stimulated) B-cells (stimulated) pSport1 H0658Ovary, Cancer 9809C332-Poorly Ovary & disease pSport1 (9809C332): Poorlydifferentiate Fallopian Tubes differentiated adenocarcinoma H0659 Ovary,Cancer Grade II Papillary Ovary disease pSport1 (15395A1F): Grade IICarcinoma, Ovary Papillary Carcinoma H0660 Ovary, Cancer: Poorlydifferentiated disease pSport1 (15799A1F) Poorly carcinoma, ovarydifferentiated carcinoma H0661 Breast, Cancer: Breast cancer diseasepSport1 (4004943 A5) H0662 Breast, Normal: Normal Breast - BreastpSport1 (4005522B2) #4005522(B2) H0663 Breast, Cancer: Breast Cancer -Breast disease pSport1 (4005522 A2) #4005522(A2) H0664 Breast, Cancer:Breast Cancer Breast disease pSport1 (9806C012R) H0665 Stromal cells3.88 Stromal cells 3.88 pSport1 H0666 Ovary, Cancer: Ovarian Cancer,Sample disease pSport1 (4004332 A2) #4004332A2 H0667 Stromal Stromalcell(HBM 3.18) pSport1 cells(HBM3.18) H0668 stromal cell clone 2.5stromal cell clone 2.5 pSport1 H0670 Ovary, Cancer(4004650 OvarianCancer - pSport1

Differentiated Micropapillary Serous Carcinoma H0671 Breast, Cancer:Breast Cancer- Sample # pSport1 (9802C02OE) 9802C02OE H0672 Ovary,Cancer: Ovarian Ovary pSport1 (4004576 A8) Cancer(4004576A8) H0673 HumanProstate Cancer, Human Prostate Cancer, Prostate Uni-ZAP XR Stage B2;re-excision stage B2 H0674 Human Prostate Cancer, Human Prostate Cancer,Prostate Uni-ZAP XR Stage C; re-excission stage C H0675 Colon, Cancer:Colon Cancer 9808C064R pCMVSport 3.0 (9808C064R) H0676 Colon, Cancer:Colon Cancer 9808C064R pCMVSport 3.0 (9808C064R)-total RNA H0677 TNFRdegenerate oligo B-Cells PCRII H0682 Serous Papillary serous papillarypCMVSport 3.0 Adenocarcinoma adenocarcinoma (9606G304SPA3B) H0683Ovarian Serous Serous papillary pCMVSport 3.0 Papillary adenocarcinoma,stage 3C Adenocarcinoma (9804G01 H0684 Serous Papillary OvarianCancer-9810G606 Ovaries pCMVSport 3.0 Adenocarcinoma H0685Adenocarcinoma of Adenocarcinoma of Ovary, pCMVSport 3.0 Ovary, HumanCell Human Cell Line, # Line, # OVCAR-3 OVCAR- H0686 Adenocarcinoma ofAdenocarcinoma of Ovary, pCMVSport 3.0 Ovary, Human Cell Human CellLine, # SW- Line 626 H0687 Human normal Human normal Ovary pCMVSport 3.0

H0688 Human Ovarian Human Ovarian pCMVSport 3.0 Cancer(#9807G017)cancer(#9807G017), mRNA from Maura Ru H0689 Ovarian Cancer OvarianCancer, pCMVSport 3.0 #9806G019 H0690 Ovarian Cancer, # Ovarian Cancer,pCMVSport 3.0 9702G001 #9702G001 H0692 BLyS Receptor from B CellLymphoma B Cell pCMVSport 3.0 Expression Cloning H0693 Normal ProstateNormal Prostate Tissue # pCMV Sport 3.0 #ODQ3958EN ODQ3958EN H0694Prostate gland Prostate gland, prostate gland pCMVSport 3.0adenocarcinoma adenocarcinoma, mod/diff, gleason H0695 mononucleocytesfrom mononucleocytes from pCMVSport 3.0 patient patient at Shady GroveHospit N0006 Human Fetal Brain Human Fetal Brain N0007 Human HippocampusHuman Hippocampus S0001 Brain frontal cortex Brain frontal cortex BrainLambda ZAP II S0002 Monocyte activated Monocyte-activated blood CellLine Uni-ZAP XR S0003 Human Osteoclastoma Osteoclastoma bone diseaseUni-ZAP XR S0005 Heart Heart-left ventricle Heart pCDNA S0007 EarlyStage Human Human Fetal Brain Uni-ZAP XR Brain S0010 Human AmygdalaAmygdala Uni-ZAP XR S0011 STROMAL - Osteoclastoma bone disease Uni-ZAPXR OSTEOCLASTOMA S0014 Kidney Cortex Kidney cortex Kidney Uni-ZAP XRS0015 Kidney medulla Kidney medulla Kidney Uni-ZAP XR S0016 KidneyPyramids Kidney pyramids Kidney Uni-ZAP XR S0022 Human OsteoclastomaOsteoclastoma Stromal Uni-ZAP XR

unamplified S0024 Human Kidney Medulla - Human Kidney Medullaunamplified S0026 Stromal cell TF274 stromal cell Bone marrow Cell LineUni-ZAP XR S0027 Smooth muscle, serum Smooth muscle Pulmanary Cell LineUni-ZAP XR treated artery S0028 Smooth muscle, control Smooth musclePulmanary Cell Line Uni-ZAP XR artery S0029 brain stem Brain stem brainUni-ZAP XR S0031 Spinal cord Spinal cord spinal cord Uni-ZAP XR S0032Smooth muscle-ILb Smooth muscle Pulmanary Cell Line Uni-ZAP XR inducedartery S0036 Human Substantia Nigra Human Substantia Nigra Uni-ZAP XRS0037 Smooth muscle, IL1b Smooth muscle Pulmanary Cell Line Uni-ZAP XRinduced artery S0038 Human Whole Brain #2 - Human Whole Brain #2 ZAPExpress Oligo dT >1.5 Kb S0040 Adipocytes Human Adipocytes from Uni-ZAPXR Osteoclastoma S0044 Prostate BPH prostate BPH Prostate diseaseUni-ZAP XR S0045 Endothelial cells-control Endothelial cell endothelialcell- Cell Line Uni-ZAP XR lung S0046 Endothelial-induced Endothelialcell endothelial cell- Cell Line Uni-ZAP XR lung S0049 Human Brain,Striatum Human Brain, Striatum Uni-ZAP XR S0050 Human Frontal Cortex,Human Frontal Cortex, disease Uni-ZAP XR Schizophrenia SchizophreniaS0051 Human Human Hypothalamus, disease Uni-ZAP XR Hypothalmus,Schizophrenia Schizophrenia S0052 neutrophils control human neutrophilsblood Cell Line Uni-ZAP XR S0053 Neutrophils IL-1 and human neutrophilinduced blood Cell Line Uni-ZAP XR

S0106 STRIATUM BRAIN disease Uni-ZAP XR DEPRESSION S0110 Brain AmygdalaBrain disease Uni-ZAP XR Depression S0112 Hypothalamus Brain Uni-ZAP XRS0114 Anergic T-cell Anergic T-cell Cell Line Uni-ZAP XR S0116 Bonemarrow Bone marrow Bone marrow Uni-ZAP XR S0118 Smooth muscle control 2Smooth muscle Pulmanary Cell Line Uni-ZAP XR artery S0126 OsteoblastsOsteoblasts Knee Cell Line Uni-ZAP XR S0132 Epithelial-TNFa and AirwayEpithelial Uni-ZAP XR INF induced S0134 Apoptotic T-cell apoptotic cellsCell Line Uni-ZAP XR S0140 eosinophil-IL5 induced eosinophil lung CellLine Uni-ZAP XR S0142 Macrophage-oxLDL macrophage-oxidized LDL bloodCell Line Uni-ZAP XR treated S0144 Macrophage (GM-CSF Macrophage (GM-CSFUni-ZAP XR treated) treated) S0146 prostate-edited prostate BPH ProstateUni-ZAP XR S0148 Normal Prostate Prostate prostate Uni-ZAP XR S0150LNCAP prostate cell LNCAP Cell Line Prostate Cell Line Uni-ZAP XR lineS0152 PC3 Prostate cell line PC3 prostate cell line Uni-ZAP XR S0176Prostate, normal, Prostate prostate Uni-ZAP XR subtraction I S0182 HumanB Cell 8866 Human B-Cell 8866 Uni-ZAP XR S0188 Prostate, BPH, Lib 2Human Prostate BPH disease pSport1 S0190 Prostate BPH, Lib 2, HumanProstate BPH pSport1 subtracted S0192 Synovial Fibroblasts SynovialFibroblasts pSport1 (control) S0194 Synovial hypoxia SynovialFibroblasts pSport1

stimulated S0206 Smooth Muscle- Smooth muscle Pulmanary Cell LinepBluescript HASTE normalized artery S0208 Messangial cell, frac 1Messangial cell pSport1 S0210 Messangial cell, frac 2 Messangial cellpSport1 S0212 Bone Marrow Stromal Bone Marrow Stromal pSport1 Cell,untreated Cell, untreated S0214 Human Osteoclastoma, Osteoclastoma bonedisease Uni-ZAP XR re-excision S0216 Neutrophils IL-1 and humanneutrophil induced blood Cell Line Uni-ZAP XR LPS induced S0218Apoptotic T-cell, re- apoptotic cells Cell Line Uni-ZAP XR excisionS0220 H. hypothalamus, frac Hypothalamus Brain ZAP Express A;re-excision S0222 H. Frontal H. Brain, Frontal Cortex, Brain diseaseUni-ZAP XR cortex, epileptic; re- Epileptic excision S0242 SynovialFibroblasts Synovial Fibroblasts pSport1 (I11/TNF), subt S0250 HumanOsteoblasts II Human Osteoblasts Femur disease pCMVSport 2.0 S0260Spinal Cord, re-excision Spinal cord spinal cord Uni-ZAP XR S0276Synovial hypoxia-RSF Synovial fobroblasts Synovial tissue pSport1subtracted (rheumatoid) S0278 H Macrophage (GM- Macrophage (GM-CSFUni-ZAP XR CSF treated), re- treated) excision S0280 Human AdiposeTissue, Human Adipose Tissue Uni-ZAP XR re-excision S0282 Brain FrontalCortex, Brain frontal cortex Brain Lambda ZAP II re-excision S0294Larynx tumor Larynx tumor Larynx, vocal disease pSport1 S0298 Bonemarrow Bone marrow Bone marrow pSport1 stroma, treated stroma, treatedSBS0300 Frontal Frontal Lobe Brain Uni-ZAP XR lobe, dementia; re-dementia/Alzheimer''s excision S0306 Larynx normal #10 261-273 Larynxnormal pSport1 S0308 Spleen/normal Spleen normal pSport1 S0310 Normaltrachea Normal trachea pSport1 S0312 Human Human osteoarthritic diseasepSport1 osteoarthritic; fraction II cartilage S0314 Human Humanosteoarthritic disease pSport1 osteoarthritis; fraction I cartilageS0318 Human Normal Human Normal Cartilage pSport1 Cartilage Fraction IIS0322 Siebben Polyposis Siebben Polyposis pSport1 S0328 Palate carcinomaPalate carcinoma Uvula disease pSport1 S0330 Palate normal Palate normalUvula pSport1 S0332 Pharynx carcinoma Pharynx carcinoma HypopharynxpSport1 S0338 Human Osteoarthritic Human osteoarthritic disease pSport1Cartilage Fracion III cartilage S0340 Human Osteoarthritic Humanosteoarthritic disease pSport1 Cartilage Fraction IV cartilage S0342Adipocytes; re-excision Human Adipocytes from Uni-ZAP XR OsteoclastomaS0344 Macrophage-oxLDL; macrophage-oxidized LDL blood Cell Line Uni-ZAPXR re-excision treated S0346 Human Amygdala; re- Amygdala Uni-ZAP XRexcision S0348 Cheek Carcinoma Cheek Carcinoma disease pSport1 S0350Pharynx Carcinoma Pharynx carcinoma Hypopharynx disease pSport1 S0352Larynx Carcinoma Larynx carcinoma disease pSport1

S0356 Colon Carcinoma Colon Carcinoma Colon disease pSport1 S0358 ColonNormal III Colon Normal Colon pSport1 S0360 Colon Tumor II Colon TumorColon disease pSport1 S0364 Human Quadriceps Quadriceps muscle pSport1S0366 Human Soleus Soleus Muscle pSport1 S0370 Larynx carcinoma IILarynx carcinoma disease pSport1 S0372 Larynx carcinoma III Larynxcarcinoma disease pSport1 S0374 Normal colon Normal colon pSport1 S0376Colon Tumor Colon Tumor disease pSport1 S0378 Pancreas normal PCA4Pancreas Normal PCA4 No pSport1 No S0380 Pancreas Tumor PCA4 PancreasTumor PCA4 Tu disease pSport1 Tu S0382 Larynx carcinoma IV Larynxcarcinoma disease pSport1 S0384 Tongue carcinoma Tongue carcinomadisease pSport1 S0388 Human Human Hypothalamus, disease Uni-ZAP XRHypothalamus, schizoph Schizophrenia renia, re-excision S0390 Smoothmuscle, control; Smooth muscle Pulmanary Cell Line Uni-ZAP XRre-excision artery S0392 Salivary Gland Salivary gland; normal pSport1S0394 Stomach; normal Stomach; normal pSport1 S0398 Testis; normalTestis; normal pSport1 S0400 Brain; normal Brain; normal pSport1 S0402Adrenal Gland, normal Adrenal gland; normal pSport1 S0404 Rectum normalRectum, normal pSport1 S0406 Rectum tumour Rectum tumour pSport1 S0408Colon, normal Colon, normal pSport1 S0410 Colon, tumour Colon, tumourpSport1 S0412 Temporal cortex- Temporal cortex, alzheimer disease OtherAlzheizmer; subtracted

Alzheimer Subtracted Subtracted S0418 CHME Cell Line; treated CHME CellLine; treated pCMVSport 3.0 5 hrs S0420 CHME Cell CHME Cell line,untreatetd pSport1 Line, untreated S0422 Mo7e Cell Line GM- Mo7e CellLine GM-CSF pCMVSport 3.0 CSF treated (1 ng/ml) treated (1 ng/ml) S0424TF-1 Cell Line GM- TF-1 Cell Line GM-CSF pSport1 CSF Treated TreatedS0426 Monocyte activated; re- Monocyte-activated blood Cell Line Uni-ZAPXR excision S0428 Neutrophils control; re- human neutrophils blood CellLine Uni-ZAP XR excision S0430 Aryepiglottis Normal Aryepiglottis NormalpSport1 S0432 Sinus piniformis Sinus piniformis Tumour pSport1 TumourS0434 Stomach Normal Stomach Normal disease pSport1 S0436 Stomach TumourStomach Tumour disease pSport1 S0438 Liver Normal Met5No Liver NormalMet5No pSport1 S0440 Liver Tumour Met 5 Tu Liver Tumour pSport1 S0442Colon Normal Colon Normal pSport1 S0444 Colon Tumor Colon Tumour diseasepSport1 S0448 Larynx Normal Larynx Normal pSport1 S0450 Larynx TumourLarynx Tumour pSport1 S0452 Thymus Thymus pSport1 S0456 Tongue NormalTongue Normal pSport1 S0458 Thyroid Normal Thyroid normal pSport1 (SDCA2No) S0460 Thyroid Tumour Thyroid Tumour pSport1 S0468 Ea.hy.926 cellline Ea.hy.926 cell line pSport1 S0472 Lung Mesothelium PYBT pSport1S0474 Human blood platelets Platelets Blood platelets Other

excission S3012 Smooth Muscle Serum Smooth muscle Pulmanary Cell LinepBluescript Treated, Norm artery S3014 Smooth muscle, serum Smoothmuscle Pulmanary Cell Line pBluescript induced, re-exc artery S6022 H.Adipose Tissue Human Adipose Tissue Uni-ZAP XR S6024 Alzheimers, spongyAlzheimer''s/Spongy Brain disease Uni-ZAP XR change change S6026 FrontalLobe, Dementia Frontal Lobe Brain Uni-ZAP XR dementia/Alzheimer''s S6028Human Manic Human Manic depression Brain disease Uni-ZAP XR DepressionTissue tissue T0002 Activated T-cells Activated T-Cell, PBL Blood CellLine pBluescript SK− fraction T0003 Human Fetal Lung Human Fetal LungpBluescript SK− T0004 Human White Fat Human White Fat pBluescript SK−T0006 Human Pineal Gland Human Pinneal Gland pBluescript SK− T0008Colorectal Tumor Colorectal Tumor disease pBluescript SK− T0010 HumanInfant Brain Human Infant Brain Other T0023 Human Pancreatic HumanPancreatic disease pBluescript SK− Carcinoma Carcinoma T0039 HSA 172Cells Human HSA172 cell line pBluescript SK− T0040 HSC172 cells SA172Cells pBluescript SK− T0041 Jurkat T-cell G1 phase Jurkat T-cellpBluescript SK− T0042 Jurkat T-Cell, S phase Jurkat T-Cell LinepBluescript SK− T0048 Human Aortic Human Aortic Endothilium pBluescriptSK− Endothelium T0049 Aorta endothelial cells + TNF-a Aorta endothelialcells pBluescript SK− T0060 Human White Adipose Human White FatpBluescript SK− T0067 Human Thyroid Human Thyroid pBluescript SK− T0069Human Uterus, normal Human Uterus, normal pBluescript SK−

T0078 Human Liver, normal Human Liver, normal Adult pBluescript SK−adult T0082 Human Adult Retina Human Adult Retina pBluescript SK− T0104HCC cell line metastisis pBluescript SK− to liver T0109 Human (HCC) cellline pBluescript SK− liver (mouse) metastasis, remake T0110 Human coloncarcinoma pBluescript SK− (HCC) cell line, remake T0112 Human (Caco-2)cell pBluescript SK− line, adenocarcinoma, colon T0114 Human (Caco-2)cell pBluescript SK− line, adenocarcinoma, colon, remake T0115 HumanColon pBluescript SK− Carcinoma (HCC) cell line L0002 Atrium cDNAlibrary Human heart L0004 ClonTech HL 1065a L0005 Clontech human aortapolyA+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult lung3″ directed MboI cDNA L0041 Human epidermal keratinocyte L0055 Humanpromyelocyte L0065 Liver HepG2 cell line. L0105 Human aorta polyA+ aorta

L0142 Human placenta cDNA placenta (TFujiwara) L0143 Human placentapolyA+ placenta (TFujiwara) L0151 Human testis (C. De testis Smet) L0157Human fetal brain brain (TFujiwara) L0158 Human fetal brain brain QBoqinL0163 Human heart cDNA heart (YNakamura) L0194 Human pancreaticpancreatic cancer Patu 8988t cancer cell line Patu 8988t L0351 Infantbrain, Bento BA, M13-derived Soares L0352 Normalized infant brain, BA,M13-derived Bento Soares L0355 P, Human foetal Brain Bluescript Wholetissue L0361 Stratagene ovary ovary Bluescript SK (#937217) L0362Stratagene ovarian Bluescript SK− cancer (#937219) L0363 NCI_CGAP_GC2germ cell tumor Bluescript SK− L0364 NCI_CGAP_GC5 germ cell tumorBluescript SK− L0366 Stratagene schizo brain schizophrenic brain S-11Bluescript SK− S11 frontal lobe L0367 NCI_CGAP_Sch1 Schwannoma tumorBluescript SK− L0368 NCI_CGAP_SS1 synovial sarcoma Bluescript SK− L0369NCI_CGAP_AA1 adrenal adenoma adrenal gland Bluescript SK−

L0371 NCI_CGAP_Br3 breast tumor breast Bluescript SK− L0372NCI_CGAP_Co12 colon tumor colon Bluescript SK− L0373 NCI_CGAP_Co11 tumorcolon Bluescript SK− L0374 NCI_CGAP_Co2 tumor colon Bluescript SK− L0375NCI_CGAP_Kid6 kidney tumor kidney Bluescript SK− L0376 NCI_CGAP_Lar1larynx larynx Bluescript SK− L0378 NCI_CGAP_Lu1 lung tumor lungBluescript SK− L0381 NCI_CGAP_HN4 squamous cell carcinoma pharynxBluescript SK− L0382 NCI_CGAP_Pr25 epithelium (cell line) prostateBluescript SK− L0383 NCI_CGAP_Pr24 invasive tumor (cell line) prostateBluescript SK− L0384 NCI_CGAP_Pr23 prostate tumor prostate BluescriptSK− L0386 NCI_CGAP_HN3 squamous cell carcinoma tongue Bluescript SK−from base of tongue L0387 NCI_CGAP_GCB0 germinal center B-cells tonsilBluescript SK− L0388 NCI_CGAP_HN6 normal gingiva (cell line BluescriptSK− from immortalized kerati L0393 B, Human Liver tissue gt11 L04111-NIB Lafmid BA L0415 b4HB3MA Cot8-HAP- Lafmid BA Ft L0435 Infant brain,LLNL lafmid BA array of Dr. M. Soares 1NIB L0438 normalized infant braintotal brain brain lafmid BA cDNA L0439 Soares infant brain whole brainLafmid BA 1NIB L0442 4HB3MK Lafmid BK L0448 3HFLSK20 Lafmid K L0455Human retina cDNA retina eye lambda gt10 randomly primed sublibrary

Tsp509I-cleaved sublibrary L0459 Adult heart, Clontech Lambda gt11 L0462WATM1 lambda gt11 L0470 BL29 Burkitt''s lambda ZAP 2 lymphoma, PascalisSideras L0471 Human fetal heart, Lambda ZAP Express Lambda ZAP ExpressL0480 Stratagene cat#937212 Lambda ZAP, (1992) pBluescript SK(−) L0481CD34+DIRECTIONAL Lambda ZAPII L0483 Human pancreatic islet Lambda ZAPIIL0485 STRATAGENE Human skeletal muscle leg muscle Lambda ZAPII skeletalmuscle cDNA library, cat. #936215. L0497 NCI_CGAP_HSC4 CD34+, CD38− frombone marrow pAMP1 normal bone marrow donor L0498 NCI_CGAP_HSC3 CD34+, Tnegative, patient bone marrow pAMP1 with chronic myelogenou L0503NCI_CGAP_Br17 adenocarcinoma breast pAMP1 L0506 NCI_CGAP_Br16 lobullarcarcinoma in situ breast pAMP1 L0511 NCI_CGAP_Ov34 borderline ovarianovary pAMP1 carcinoma L0512 NCI_CGAP_Ov36 borderline ovarian ovary pAMP1carcinoma L0515 NCI_CGAP_Ov32 papillary serous carcinoma ovary pAMP1L0517 NCI_CGAP_Pr1 pAMP10 L0518 NCI_CGAP_Pr2 pAMP10 L0519 NCI_CGAP_Pr3pAMP10 L0520 NCI_CGAP_Alv1 alveolar pAMP10 rhabdomyosarcoma

L0522 NCI_CGAP_Kid1 kidney pAMP10 L0523 NCI_CGAP_Lip2 liposarcoma pAMP10L0525 NCI_CGAP_Li2 liver pAMP10 L0526 NCI_CGAP_Pr12 metastatic prostatebone pAMP10 lesion L0527 NCI_CGAP_Ov2 ovary pAMP10 L0529 NCI_CGAP_Pr6prostate pAMP10 L0532 NCI_CGAP_Thy1 thyroid pAMP10 L0534 Chromosome 7Fetal brain brain pAMP10 Brain cDNA Library L0540 NCI_CGAP_Pr10 invasiveprostate tumor prostate pAMP10 L0542 NCI_CGAP_Pr11 normal prostaticepithelial prostate pAMP10 cells L0543 NCI_CGAP_Pr9 normal prostaticepithelial prostate pAMP10 cells L0544 NCI_CGAP_Pr4 prostaticintraepithelial prostate pAMP10 neoplasia - high grade L0545NCI_CGAP_Pr4.1 prostatic intraepithelial prostate pAMP10 neoplasia -high grade L0549 NCI_CGAP_HN10 carcinoma in situ from pAMP10 retromolartrigone L0551 NCI_CCAP_HN7 normal squamous pAMP10 epithelium, floor ofmouth L0555 NCI_CGAP_Lu34 large cell carcinoma lung pAMP10 L0561NCI_CGAP_HN11 normal squamous tongue pAMP10 epithelium L0562 Chromosome7 HeLa HeLa cell line; pAMP10 cDNA Library ATCC L0563 Human Bone Marrowbone marrow pBluescript Stromal Fibroblast L0564 Jia bone marrow stromabone marrow stroma pBluescript L0565 Normal Human Bone Hip pBluescript

L0581 Stratagene liver liver pBluescript SK (#937224) L0584 StratagenecDNA pBluescript SK(+) library Human heart, cat#936208 L0586 HTCDL1pBluescript SK(−) L0588 Stratagene endothelial pBluescript SK− cell937223 L0589 Stratagene fetal retina pBluescript SK− 937202 L0590Stratagene fibroblast pBluescript SK− (#937212) L0591 Stratagene HeLacell s3 pBluescript SK− 937216 L0592 Stratagene hNT neuron pBluescriptSK− (#937233) L0593 Stratagene pBluescript SK− neuroepithelium (#937231)L0594 Stratagene pBluescript SK− neuroepithelium NT2RAMI 937234 L0595Stratagene NT2 neuroepithelial cells brain pBluescript SK− neuronalprecursor 937230 L0596 Stratagene colon colon pBluescript SK− (#937204)L0597 Stratagene corneal cornea pBluescript SK− stroma (#937222) L0598Morton Fetal Cochlea cochlea ear pBluescript SK− L0599 Stratagene lunglung pBluescript SK− (#937210)

Epithelium L0601 Stratagene pancreas pancreas pBluescript SK− (#937208)L0602 Pancreatic Islet pancreatic islet pancreas pBluescript SK− L0603Stratagene placenta placenta pBluescript SK− (#937225) L0604 Stratagenemuscle muscle skeletal muscle pBluescript SK− 937209 L0605 Stratagenefetal spleen fetal spleen spleen pBluescript SK− (#937205) L0606NCI_CGAP_Lym5 follicular lymphoma lymph node pBluescript SK− L0607NCI_CGAP_Lym6 mantle cell lymphoma lymph node pBluescript SK− L0608Stratagene lung lung carcinoma lung NCI-H69 pBluescript SK− carcinoma937218 L0611 Schiller meningioma meningioma brain pBluescript SK−(Stratagene) L0612 Schiller oligodendroglioma brain pBluescript SK−oligodendroglioma (Stratagene) L0615 22 week old human fetalpBluescriptII SK(−) liver cDNA library L0617 Chromosome 22 exonpBluescriptIIKS+ L0622 HM1 pcDNAII (Invitrogen) L0623 HM3 pectoralmuscle (after pcDNAII (Invitrogen) mastectomy) L0625 NCI_CGAP_AR1 bulkalveolar tumor pCMV-SPORT2 L0626 NCI_CGAP_GC1 bulk germ cell seminomapCMV-SPORT2 L0627 NCI_CGAP_Co1 bulk tumor colon pCMV-SPORT2 L0629NCI_CGAP_Mel3 metastatic melanoma to bowel (skin pCMV-SPORT4 bowelprimary) L0631 NCI_CGAP_Br7 breast pCMV-SPORT4 L0634 NCI_CGAP_Ov8 serousadenocarcinoma ovary pCMV-SPORT4 L0635 NCI_CGAP_PNS1 dorsal rootganglion peripheral pCMV-SPORT4 L0636 NCI_CGAP_Pit1 four pooledpituitary brain pCMV-SPORT6 adenomas L0637 NCI_CGAP_Brn53 three pooledmeningiomas brain pCMV-SPORT6 L0638 NCI_CGAP_Brn35 tumor, 5 pooled (seebrain pCMV-SPORT6 description) L0639 NCI_CGAP_Brn52 tumor, 5 pooled (seebrain pCMV-SPORT6 description) L0640 NCI_CGAP_Br18 four pooledhigh-grade breast pCMV-SPORT6 tumors, including two prima L0641NCI_CGAP_Co17 juvenile granulosa tumor colon pCMV-SPORT6 L0642NCI_CGAP_Co18 moderately differentiated colon pCMV-SPORT6 adenocarcinomaL0643 NCI_CGAP_Co19 moderately differentiated colon pCMV-SPORT6adenocarcinoma L0644 NCI_CGAP_Co20 moderately differentiated colonpCMV-SPORT6 adenocarcinoma L0645 NCI_CGAP_Co21 moderately differentiatedcolon pCMV-SPORT6 adenocarcinoma L0646 NCI_CGAP_Co14moderately-differentiated colon pCMV-SPORT6 adenocarcinoma L0647NCI_CGAP_Sar4 five pooled sarcomas, connective pCMV-SPORT6 includingmyxoid tissue liposarcoma L0648 NCI_CGAP_Eso2 squamous cell carcinomaesophagus pCMV-SPORT6 L0649 NCI_CGAP_GU1 2 pooled high-gradegenitourinary pCMV-SPORT6 transitional cell tumors tract L0650NCI_CGAP_Kid13 2 pooled Wilms'' tumors, kidney pCMV-SPORT6 one primaryand one metast L0651 NCI_CGAP_Kid8 renal cell tumor kidney pCMV-SPORT6L0652 NCI_CGAP_Lu27 four pooled poorly- lung pCMV-SPORT6 differentiatedL0653 NCI_CGAP_Lu28 two pooled squamous cell lung pCMV-SPORT6 carcinomasL0654 NCI_CGAP_Lu31 lung, cell line pCMV-SPORT6 L0655 NCI_CGAP_Lym12lymphoma, follicular mixed lymph node pCMV-SPORT6 small and large cellL0656 NCI_CGAP_Ov38 normal epithelium ovary pCMV-SPORT6 L0657NCI_CGAP_Ov23 tumor, 5 pooled (see ovary pCMV-SPORT6 description) L0658NCI_CGAP_Ov35 tumor, 5 pooled (see ovary pCMV-SPORT6 description) L0659NCI_CGAP_Pan1 adenocarcinoma pancreas pCMV-SPORT6 L0661 NCI_CGAP_Mel15malignant melanoma, skin pCMV-SPORT6 metastatic to lymph node L0662NCI_CGAP_Gas4 poorly differentiated stomach pCMV-SPORT6 adenocarcinomawith signet r L0663 NCI_CGAP_Ut2 moderately-differentiated uteruspCMV-SPORT6 endometrial adenocarcino L0664 NCI_CGAP_Ut3poorly-differentiated uterus pCMV-SPORT6 endometrial adenocarcinoma,L0665 NCI_CGAP_Ut4 serous papillary carcinoma, uterus pCMV-SPORT6 highgrade, 2 pooled t L0666 NCI_CGAP_Ut1 well-differentiated uteruspCMV-SPORT6 endometrial adenocarcinoma, 7 L0667 NCI_CGAP_CML1 myeloidcells, 18 pooled whole blood pCMV-SPORT6 CML cases, BCR/ABL rearra L0686Stanley Frontal SN pool 2 frontal lobe (see brain pCR2.1-TOPOdescription) (Invitrogen) L0698 Testis 2 PGEM 5 zf(+)

hncDNA library L0709 NIH_MGC_21 choriocarcinoma placenta pOTB7 L0710NIH_MGC_7 small cell carcinoma lung MGC3 pOTB7 L0717 Gessler Wilms tumorpSPORT1 L0718 Testis 5 pSPORT1 L0731 Soares_pregnant_uterus_(—) uteruspT7T3-Pac NbHPU L0738 Human colorectal pT7T3D cancer L0740 Soaresmelanocyte melanocyte pT7T3D (Pharmacia) 2NbHM with a modifiedpolylinker L0741 Soares adult brain brain pT7T3D (Pharmacia) N2b4HB55Ywith a modified polylinker L0742 Soares adult brain brain pT7T3D(Pharmacia) N2b5HB55Y with a modified polylinker L0743 Soares breast2NbHBst breast pT7T3D (Pharmacia) with a modified polylinker L0744Soares breast 3NbHBst breast pT7T3D (Pharmacia) with a modifiedpolylinker L0745 Soares retina N2b4HR retina eye pT7T3D (Pharmacia) witha modified polylinker L0746 Soares retina N2b5HR retina eye pT7T3D(Pharmacia) with a modified polylinker L0747 Soares_fetal_heart_NbHH19Wheart pT7T3D (Pharmacia) with a modified

L0748 Soares fetal liver spleen Liver and pT7T3D (Pharmacia) 1NFLSSpleen with a modified polylinker L0749 Soares_fetal_liver_spleen_(—)Liver and pT7T3D (Pharmacia) 1NFLS_S1 Spleen with a modified polylinkerL0750 Soares_fetal_lung_NbHL19W lung pT7T3D (Pharmacia) with a modifiedpolylinker L0751 Soares ovary tumor ovarian tumor ovary pT7T3D(Pharmacia) NbHOT with a modified polylinker L0752Soares_parathyroid_tumor_(—) parathyroid tumor parathyroid pT7T3D(Pharmacia) NbHPA gland with a modified polylinker L0753Soares_pineal_gland_N3HPG pineal gland pT7T3D (Pharmacia) with amodified polylinker L0754 Soares placenta Nb2HP placenta pT7T3D(Pharmacia) with a modified polylinker L0755Soares_placenta_8to9weeks_(—) placenta pT7T3D (Pharmacia) 2NbHP8to9Wwith a modified polylinker L0756 Soares_multiple_sclerosis_(—) multiplesclerosis lesions pT7T3D (Pharmacia) 2NbHMSP with a modified polylinkerV_TYPE L0757 Soares_senescent_fibroblasts_(—) senescent fibroblastpT7T3D (Pharmacia) NbHSF with a modified polylinker V_TYPE L0758Soares_testis_NHT pT7T3D-Pac (Pharmacia) with a

L0759 Soares_total_fetus_Nb2HF8_(—) pT7T3D-Pac 9w (Pharmacia) with amodified polylinker L0760 Barstead aorta HPLRB3 aorta pT7T3D-Pac(Pharmacia) with a modified polylinker L0761 NCI_CGAP_CLL1 B-cell,chronic lymphotic pT7T3D-Pac leukemia (Pharmacia) with a modifiedpolylinker L0762 NCI_CGAP_Br1.1 breast pT7T3D-Pac (Pharmacia) with amodified polylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia)with a modified polylinker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac(Pharmacia) with a modified polylinker L0765 NCI_CGAP_Co4 colonpT7T3D-Pac (Pharmacia) with a modified polylinker L0766 NCI_CGAP_GCB1germinal center B cell pT7T3D-Pac (Pharmacia) with a modified polylinkerL0767 NCI_CGAP_GC3 pooled germ cell tumors pT7T3D-Pac (Pharmacia) with amodified polylinker L0768 NCI_CGAP_GC4 pooled germ cell tumorspT7T3D-Pac (Pharmacia) with a modified polylinker L0769 NCI_CGAP_Brn25anaplastic brain pT7T3D-Pac oligodendroglioma (Pharmacia) with a

L0770 NCI_CGAP_Brn23 glioblastoma (pooled) brain pT7T3D-Pac (Pharmacia)with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinoma colonpT7T3D-Pac (Pharmacia) with a modified polylinker L0772 NCI_CGAP_Co10colon tumor RER+ colon pT7T3D-Pac (Pharmacia) with a modified polylinkerL0773 NCI_CGAP_Co9 colon tumor RER+ colon pT7T3D-Pac (Pharmacia) with amodified polylinker L0774 NCI_CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia)with a modified polylinker L0775 NCI_CGAP_Kid5 2 pooled tumors (clearcell kidney pT7T3D-Pac type) (Pharmacia) with a modified polylinkerL0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0777 Soares_NhHMPu_S1 Pooled human melanocyte, mixed (seepT7T3D-Pac fetal heart, and pregnant below) (Pharmacia) with a modifiedpolylinker L0779 Soares_NFL_T_GBC_S1 pooled pT7T3D-Pac (Pharmacia) witha modified polylinker L0780 Soares_NSF_F8_9W_O pooled pT7T3D-PacT_PA_P_S1 (Pharmacia) with a modified polylinker L0782 NCI_CGAP_Pr21normal prostate prostate pT7T3D-Pac (Pharmacia) with a

L0783 NCI_CGAP_Pr22 normal prostate prostate pT7T3D-Pac (Pharmacia) witha modified polylinker L0784 NCI_CGAP_Lei2 leiomyosarcoma soft tissuepT7T3D-Pac (Pharmacia) with a modified polylinker L0785 Barstead spleenspleen pT7T3D-Pac HPLRB2 (Pharmacia) with a modified polylinker L0786Soares_NbHFB whole brain pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0787 NCI_CGAP_Sub1 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0788 NCI_CGAP_Sub2 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0789 NCI_CGAP_Sub3 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0790 NCI_CGAP_Sub4 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0791 NCI_CGAP_Sub5 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0792 NCI_CGAP_Sub6 pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0793 NCI_CGAP_Sub7 pT7T3D-Pac (Pharmacia) with a

L0794 NCI_CGAP_GC6 pooled germ cell tumors pT7T3D-Pac (Pharmacia) with amodified polylinker L0796 NCI_CGAP_Brn50 medulloblastoma brainpT7T3D-Pac (Pharmacia) with a modified polylinker L0800 NCI_CGAP_Co16colon tumor, RER+ colon pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0803 NCI_CGAP_Kid11 kidney pT7T3D-Pac (Pharmacia) with amodified polylinker L0804 NCI_CGAP_Kid12 2 pooled tumors (clear cellkidney pT7T3D-Pac type) (Pharmacia) with a modified polylinker L0805NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0806 NCI_CGAP_Lu19 squamous cell carcinoma, lung pT7T3D-Pacpoorly differentiated (4 (Pharmacia) with a modified polylinker L0807NCI_CGAP_Ov18 fibrotheoma ovary pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0808 Barstead prostate BPH prostate pT7T3D-Pac HPLRB4 1(Pharmacia) with a modified polylinker L0809 NCI_CGAP_Pr28 prostatepT7T3D-Pac (Pharmacia) with a modified polylinker L1430 CT0225 colonpuc18 L1562 CN0027 colon_normal puc18

L2255 GLC corresponding non pBluescript sk(−) cancerous liver tissueL2257 NIH_MGC_65 adenocarcinoma colon pCMV-SPORT6 L2258 NIH_MGC_67retinoblastoma eye pCMV-SPORT6 L2259 NIH_MGC_68 large cell carcinomalung pCMV-SPORT6 L2260 NIH_MGC_69 large cell carcinoma, lung pCMV-SPORT6undifferentiated L2261 NIH_MGC_70 epithelioid carcinoma pancreaspCMV-SPORT6 L2262 NIH_MGC_72 melanotic melanoma skin pCMV-SPORT6 L2263NIH_MGC_66 adenocarcinoma ovary pCMV-SPORT6 L2264 NIH_MGC_71leiomyosarcoma uterus pCMV-SPORT6 L2270 Lupski_dorsal_root_gangliondorsal root ganglia pCMV-SPORT6 (Life Technologies) L2285 BT0723 breastpuc18 L2293 BT0762 breast puc18 L2300 BT0789 breast puc18 L2323 CT0406colon puc18 L2359 UT0023 uterus_tumor puc18 L2368 UT0041 uterus_tumorpuc18 L2402 NN0118 nervous_normal puc18 L2482 HT0497 head_neck puc18L2486 HT0527 head_neck puc18 L2491 HT0559 head_neck puc18 L2497 HT0618head_neck puc18 L2499 HT0622 head_neck puc18 L2528 HT0713 head_neckpuc18 L2630 HT0865 head_neck puc18 L2647 HT0894 head_neck puc18 L2652NIH_MGC_57 glioblastoma brain pDNR-LIB (Clontech) L2653 NIH_MGC_58hypernephroma kidney pDNR-LIB (Clontech) L2654 NIH_MGC_9 adenocarcinomacell line ovary pOTB7

leukemia L2657 NIH_MGC_54 from chronic myelogenous bone marrow pDNR-LIB(Clontech) leukemia L2670 NT0023 nervous_tumor puc18 L2744 FT0004prostate_tumor puc18 L2759 FT0028 prostate_tumor puc18 L2777 FT0056prostate_tumor puc18 L2800 FT0097 prostate_tumor puc18 L2879 AN0032amnion_normal puc18 L2903 BN0039 breast_normal puc18 L2906 BN0047breast_normal puc18 L2909 BN0067 breast_normal puc18 L2910 BN0070breast_normal puc18 L3012 BN0296 breast_normal puc18 L3109 ET0046lung_tumor puc18 13180 MT0101 marrow puc18 L3181 MT0107 marrow puc18L3215 OT0083 ovary puc18 L3271 FN0094 prostate_normal puc18 L3278 FN0104prostate_normal puc18 L3311 FN0180 prostate_normal puc18 L3387 GKBhepatocellular carcinoma pBluescript sk(−) L3388 GKC hepatocellularcarcinoma pBluescript sk(−) L3391 NIH_MGC_53 carcinoma, cell linebladder pDNR-LIB (Clontech) L3485 GN0070 placenta_normal puc18 L3499HT0617 head_neck puc18 L3503 HT0870 head_neck puc18 L3576 TN0086testis_normal puc18 L3643 ADB Adrenal gland pBluescript sk(−) L3644 ADCAdrenal gland pBluescript sk(−) L3645 Cu adrenal cortico adenoma forpBluescript sk(−) Cushing''s syndrome L3646 DCA pTriplEx2 L3647 HumanHO-1 melanoma cells L3649 DCB pTriplEx2 L3652 FHTB hypothalamuspTriplEx2 L3653 HTB Hypothalamus pBluescript sk(−) L3655 HTCHypothalamus pBluescript sk(−) L3657 HTF Hypothalamus pBluescript sk(−)L3658 cdA pheochromocytoma pTriplEx2 L3659 CB cord blood pBluescriptL3663 NIH_MGC_60 adenocarcinoma prostate pDNR-LIB (Clontech) L3665NIH_MGC_75 kidney pDNR-LIB (Clontech) L3709 CT0515 colon puc18 L3726GN0038 placenta_normal puc18 L3796 UT0042 uterus_tumor puc18 L3811 NPCpituitary pBluescript sk(−) L3814 BM Bone marrow pTriplEx2 L3815 MDSBone marrow pTriplEx2 L3816 HEMBA1 whole embryo, mainly head pME18SFL3L3817 HEMBB1 whole embryo, mainly body pME18SFL3 L3818 MAMMA1 mammarygland pME18SFL3 L3821 NIH_MGC_48 primary B-cells from B-cells pOTB7tonsils (cell line) L3822 NIH_MGC_59 mucoepidermoid carcinoma lungpDNR-LIB (Clontech) L3824 NT2RM2 NT2 pME18SFL3 L3825 NT2RM4 NT2pME18SFL3 L3826 NT2RP1 NT2 pUC19FL3 L3827 NT2RP2 NT2 pME18SFL3 L3828NT2RP3 NT2 pME18SFL3 L3829 NT2RP4 NT2 pME18SFL3 L3831 OVARC1 ovary,tumor tissue pME18SFL3 L3832 PLACE1 placenta pME18SFL3 L3833 PLACE2placenta pME18SFL3 L3834 PLACE3 placenta pME18SFL3 L3837 THYRO1 thyroidgland pME18SFL3 L3872 NCI_CGAP_Skn1 skin, normal, 4 pCMV-SPORT6 pooledsa L3904 NCI_CGAP_Brn64 glioblastoma with EGFR brain pCMV-SPORT6amplification L3905 NCI_CGAP_Brn67 anaplastic brain pCMV-SPORT6oligodendroglioma with 1p/19q loss L4497 NCI_CGAP_Br22 invasive ductalcarcinoma, breast pCMV-SPORT6 3 pooled samples L4501 NCI_CGAP_Sub8pT7T3D-Pac (Pharmacia) with a modified polylinker L4508 NCI_CGAP_Thy8normal epithelium thyroid pAMP10 L4556 NCI_CGAP_HN13 squamous cellcarcinoma tongue pCMV-SPORT6 L4558 NCI_CGAP_Pan3 pancreas pCMV-SPORT6L4559 NCI_CGAP_Thy3 follicular carcinoma thyroid pCMV-SPORT6 L4747NCI_CGAP_Brn41 oligodendroglioma brain pT7T3D-Pac (Pharmacia) with amodified polylinker L5564 NCI_CGAP_HN20 normal pAMP1 head/neck tissueL5565 NCI_CGAP_Brn66 glioblastoma with probably brain pCMV-SPORT6 TP53mutation and witho L5566 NCI_CGAP_Brn70 anaplastic brainpCMV-SPORT6.ccdb oligodendroglioma L5572 NCI_CGAP_Co27 adenocarcinoma(mucinous colon pAMP1 component) L5574 NCI_CGAP_HN19 normal epitheliumnasopharynx pAMP10 L5575 NCI_CGAP_Brn65 glioblastoma without EGFR brainpCMV-SPORT6 amplification L5622 NCI_CGAP_Skn3 skin pCMV-SPORT6 L5623NCI_CGAP_Skn4 squamous cell carcinoma skin pCMV-SPORT6Description of Table 5

Table 5 provides a key to the OMIM reference identification numbersdisclosed in Table 1B.1, column 9. OMIM reference identification numbers(Column 1) were derived from Online Mendelian Inheritance in Man (OnlineMendelian Inheritance in Man, OMIM. McKusick-Nathans Institute forGenetic Medicine, Johns Hopkins University (Baltimore, Md.) and NationalBiotechnology Information, National Library of Medicine, (Bethesda, Md.)2000. Web URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 providesdiseases with the cytologic band disclosed in Table 1B.1, column 8, asdetermined using the database. TABLE 5 OMIM Reference Description 100690Myasthenic syndrome, slow-channel congenital, 601462 100710 Myasthenicsyndrome, slow-channel congenital, 601462 100730 Myasthenia gravis,neonatal transient 102200 Somatotrophinonia 102700 Severe combinedimmunodeficiency due to ADA deficiency 102700 Hemolytic anemia due toADA excess 103581 Albright hereditary osteodystrophy-2 104770Amyloidosis, secondary, susceptibility to 106100 Angioedema, hereditary106150 Hypertension, essential, susceptibility to 106150 Preeclampsia,susceptibility to 106165 Hypertension, essential, 145500 107250 Anteriorsegment mesenchymal dysgenesis 107300 Antithrombin III deficiency 107670Apolipoprotein A-II deficiency 108725 Atherosclerosis, susceptibility to108985 Atrophia areata 109270 Renal tubular acidosis, distal, 179800109270 Spherocytosis, hereditary 109270 [Acanthocytosis, one form]109270 [Elliptocytosis, Malaysian-Melanesian type] 109270 Hemolyticanemia due to band 3 defect 110100 Blepharophimosis, epicanthusinversus, and ptosis, type 1 110700 Vivax malaria, susceptibility to112410 Hypertension with brachydactyly 116806 Colorectal cancer 116860Cavernous angiomatous malformations 117700 [Hypoceruloplasminemia,hereditary] 117700 Hemosiderosis, systemic, due to aceruloplasminemia118485 Polycystic ovary syndrome with hyperandrogenemia 118800Choreoathetosis, familial paroxysmal 120070 Alport syndrome, autosomalrecessive, 203780 120120 Epidermolysis bullosa dystrophica, dominant,131750 120120 Epidermolysis bullosa dystrophica, recessive, 226600120120 Epidermolysis bullosa, pretibial, 131850 120131 Alport syndrome,autosomal recessive, 203780 120131 Hematuria, familial benign 120150Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420,166220 120150 Osteoporosis, idiopathic, 166710 120150 Ehlers-Danlossyndrome, type VIIA1, 130060 120436 Muir-Torre family cancer syndrome,158320 120436 Turcot syndrome with glioblastoma, 276300 120436Colorectal cancer, hereditary nonpolyposis, type 2 120550 C1qdeficiency, type A 120570 C1q deficiency, type B 120575 C1q deficiency,type C 120700 C3 deficiency 123000 Craniometaphyseal dysplasia 123620Cataract, cerulean, type 2, 601547 123660 Cataract, Coppock-like 129900EEC syndrome-1 130500 Elliptocytosis-1 131100 Multiple endocrineneoplasia I 131100 Prolactinoma, hyperparathyroidism, carcinoid syndrome131100 Carcinoid tumor of lung 131210 Atherosclerosis, susceptibility to133171 [Erythrocytosis, familial], 133100 133200 Erythrokeratodermiavariabilis 133450 Neuroepithelioma 133450 Ewing sarcoma 133701Exostoses, multiple, type 2 133780 Vitreoretinopathy, exudative,familial 134790 Hyperferritinemia-cataract syndrome, 600886 134820Dysfibrinogenemia, alpha type, causing bleeding diathesis 134820Dysfibrinogenemia, alpha type, causing recurrent thrombosis 134820Amyloidosis, hereditary renal, 105200 134830 Dysfibrinogenemia, betatype 134850 Dysfibrinogenemia, gamma type 134850 Hypofibrinogenemia,gamma type 135600 Ehlers-Danlos syndrome, type X 135700 Fibrosis ofextraocular muscles, congenital, 1 135940 Ichthyosis vulgaris, 146700136132 [Fish-odor syndrome], 602079 136836 Fucosyltransferase-6deficiency 138030 [Hyperproglucagonemia] 138140 Glucose transportdefect, blood-brain barrier 138190 Diabetes mellitus,noninsulin-dependent 138300 Hemolytic anemia due to glutathionereductase deficiency 138320 Hemolytic anemia due to glutathioneperoxidase deficiency 141750 Alpha-thalassemia/mental retardationsyndrome, type 1 141800 Methemoglobinemias, alpha- 141800 Thalassemias,alpha- 141800 Erythremias, alpha- 141800 Heinz body anemias, alpha-141850 Thalassemia, alpha- 141850 Erythrocytosis 141850 Heinz bodyanemia 141850 Hemoglobin H disease 141850 Hypochromic microcytic anemia142989 Synpolydactyly, type II, 186000 145001 Hyperparathyroidism-jawtumor syndrome i45260 Pseudohypoaldosteronism, type II 145981Hypocalciuric hypercalcemia, type II 146150 Hypomelanosis of Ito 146790Lupus nephritis, susceptibility to 147050 Atopy 147141 Leukemia, acutelymphoblastic 147200 [Kappa light chain deficiency] 148065 White spongenevus, 193900 148080 Epidermolytic hyperkeratosis, 113800 148370Keratolytic winter erythema 150210 Lactoferrin-deficient neutrophils,245480 151410 Leukemia, chronic myeloid 152445 Vohwinkel syndrome,124500 152445 Erythrokeratoderma, progressive symmetric, 602036 152760Hypogonadotropic hypogonadism due to GNRH deficiency, 227200 153454Ehiers-Danlos syndrome, type VI, 225400 153700 Macular dystrophy,vitelliform type 154275 Malignant hyperthermia susceptibility 2 156232Mesomelic dysplasia, Kantaputra type 156850 Cataract, congenital, withmicrophthalmia 157147 Abetalipoproteinemia, 200100 157640 PEO withmitochondrial DNA deletions, type 1 157655 Lactic acidosis due to defectin iron-sulfur cluster of complex I 159001 Muscular dystrophy,limb-girdle, type 1B 161015 Mitochondrial complex I deficiency, 252010164009 Leukemia, acute promyelocytic, NUMA/RARA type 164953 Liposarcoma168360 Paraneoplastic sensory neuropathy 168461 Multiple myeloma, 254250168461 Parathyroid adenomatosis 1 168461 Centrocytic lymphoma 168468Metaphyseal chondrodysplasia, Murk Jansen type, 156400 168470 Humoralhypercalcemia of malignancy 168500 Parietal foramina 169600Hailey-Hailey disease 171190 Hypertension, essential, 145500 171650Lysosomal acid phosphatase deficiency 171760 Hypophosphatasia, adult,146300 171760 Hypophosphatasia, infantile, 241500 173610 Plateletalpha/delta storage pooi deficiency 173870 Xeroderma pigmentosum 173870Fanconi anemia 174000 Medullary cystic kidney disease, AD 174900Polyposis, juvenile intestinal 176100 Porphyria cutanea tarda 176100Porphyria, hepatoerythropoietic 176830 Obesity, adrenal insufficiency,and red hair 176830 ACTH deficiency 176930 Dysprothrombinemia 176930Hypoprothrombinemia 176943 Apert syndrome, 101200 176943 Pfeiffersyndrome, 101600 176943 Beare-Stevenson cutis gyrata syndrome, 123790176943 Crouzon craniofacial dysostosis, 123500 176943 Jackson-Weisssyndrome, 123150 177070 Spherocytosis, hereditary, Japanese type 177070Hermansky-Pudlak syndrome, 203300 178300 Ptosis, hereditary congenital,1 178600 Pulmonary hypertension, familial primary 178640 Pulmonaryalveolar proteinosis, congenital, 265120 179755 Renal cell carcinoma,papillary, 1 180100 Retinitis pigmentosa-1 180380 Night blindness,congenital stationery, rhodopsin-related 180380 Retinitis pigmentosa,autosomal recessive 180380 Retinitis pigmentosa-4, autosomal dominant180721 Retinitis pigmentosa, digenic 180840 Susceptibility to IDDM181405 Scapuloperoneal spinal muscular atrophy, New England type 181430Scapuloperoneal syndrome, myopathic type 181600 Sclerotylosis 182138Anxiety-related personality traits 182279 Prader-Willi syndrome 182280Small-cell cancer of lung 182500 Cataract, congenital 182601 Spasticparaplegia-4 182860 Pyropoikilocytosis 182860 Spherocytosis, recessive182860 Elliptocytosis-2 185430 Atherosclerosis, susceptibility to 185800Symphalangism, proximal 186580 Arthrocutaneouveal granulomatosis 186860Leukemia/lymphoma, T-cell 186921 Leukemia, T-cell acute lymphoblastic188070 Bleeding disorder due to defective thromboxane A2 receptor 188450Goiter, adolescent multinodular 188450 Goiter, nonendemic, simple 188450Hypothyroidism, hereditary congenital 188826 Sorsby fundus dystrophy,136900 189800 Preeclampsia/eclampsia 191092 Tuberous sclerosis-2 191170Colorectal cancer, 114500 191170 Li-Fraumeni syndrome 191181 Cervicalcarcinoma 191315 Insensitivity to pain, congenital, with anhidrosis,256800 192340 Diabetes insipidus, neurohypophyseal, 125700 193235Vitreoretinopathy, neovascular inflammatory 200990 Acrocallosal syndrome201460 Acyl-CoA dehydrogenase, long chain, deficiency of 203100Waardenburg syndrome/ocular albinism, digenic, 103470 203100 Albinism,oculocutaneous, type IA 203200 Albinism, ocular, autosomal recessive203200 Albinism, oculocutaneous, type II 203500 Alkaptonuria 203800Alstrom syndrome 205100 Amyotrophic lateral sclerosis, juvenile 209901Bardet-Biedl syndrome 1 214500 Chediak-Higashi syndrome 216900Achromatopsia 218000 Andermann syndrome 221820 Gliosis, familialprogressive subcortical 227220 [Eye color, brown] 227646 Fanconi anemia,type D 229800 [Fructosuria] 230000 Fucosidosis 230800 Gaucher disease230800 Gaucher disease with cardiovascular calcification 231680Glutaricaciduria, type IIA 232050 Propionicacidemia, type II or pccBtype 232600 McArdle disease 233700 Chronic granulomatous disease due todeficiency of NCF-1 234200 Neurodegeneration with brain ironaccumulation 236730 Urofacial syndrome 238600 Chylomicronemia syndrome,familial 238600 Combined hyperlipemia, familial 238600Hyperlipoproteinemia I 238600 Lipoprotein lipase deficiency 240400Scurvy 243500 Isovalericacidemia 245000 Papillon-Lefevre syndrome 248510Mannosidosis, beta- 248611 Maple syrup urine disease, type Ib 249000Meckel syndrome 249270 Thiamine-responsive megaloblastic anemia 253250Mulibrey nanism 254210 Myasthenia gravis, familial infantile 255800Schwartz-Jampel syndrome 256700 Neuroblastoma 258870 Gyrate atrophy ofchoroid and retina with omithinemia, B6 responsive or unresponsive259700 Osteopetrosis, recessive 259770 Osteoporosis-pseudogliomasyndrome 259900 Hyperoxaluria, primary, type 1 261510 Pseudo-Zellwegersyndrome 262000 Bjornstad syndrome 263700 Porphyria, congenitalerythropoietic 266150 Pyruvate carboxylase deficiency 266200 Anemia,hemolytic, due to PK deficiency 266300 [Hair color, red] 271900 Canavandisease 272800 Tay-Sachs disease 272800 [Hex A pseudodeficiency] 272800GM2-gangliosidosis, juvenile, adult 276700 Tyrosinemia, type I 276902Usher syndrome, type 3 276903 Usher syndrome, type 1B 276903 Deafness,autosomal dominant 11, neurosensory, 601317 276903 Deafness, autosomalrecessive 2, neurosensory, 600060 278250 Wrinkly skin syndrome 600040Colorectal cancer 600045 Xeroderma pigmentosum, group E, subtype 2600079 Colon cancer 600119 Muscular dystrophy, Duchenne-like, type 2600119 Adhalinopathy, primary 600140 Rubenstein-Taybi syndrome, 180849600143 Epilepsy, progressive, with mental retardation 600163 Long QTsyndrome-3 600179 Leber congenital amaurosis, type I, 204000 600194Ichthyosis bullosa of Siemens, 146800 600231 Palmoplantar keratoderma,Bothnia type 600258 Colorectal cancer, hereditary nonpolyposis, type 3600273 Polycystic kidney disease, infantile severe, with tuberoussclerosis 600281 Non-insulin-dependent diabetes mellitus, 125853 600281MODY, type 1, 125850 600319 Diabetes mellitus, insulin-dependent, 4600512 Epilepsy, partial 600525 Trichodontoosseous syndrome, 190320600528 CPT deficiency, hepatic, type I, 255120 600623 Prostate cancer,176807 600698 Salivary adenoma 600698 Uterine leiomyoma 600698 Lipoma600698 Lipomatosis, mutiple, 151900 600759 Alzheimer disease-4 600808Enuresis, nocturnal, 2 600811 Xeroderma pigmentosum, group E,DDB-negative subtype, 278740 600839 Bartter syndrome, 241200 600850Schizophrenia disorder-4 600881 Cataract, congenital, zonular, withsutural opacities 600882 Charcot-Marie-Tooth neuropathy-2B 600897Cataract, zonular pulverulent-1, 116200 600957 Persistent Mullerian ductsyndrome, type I, 261550 600958 Cardiomyopathy, familial hypertrophic,4, 115197 600977 Cone dystrophy, progressive 600983Pseudohypoaldosteronism type I, autosomal dominant, 177735 600996Arrhythmogenic right ventricular dysplasia-2 601105 Pycnodysostosis,265800 601154 Cardiomyopathy, dilated, 1E 601199 Neonatalhyperparathyroidism, 239200 601199 Hypocalcemia, autosomal dominant,601198 601199 Hypocalciuric hypercalcemia, type I, 145980 601202Cataract, anterior olar-2 601238 Cerebellar ataxia, Cayman type 601277Ichthyosis, lamellar, type 2 601284 Hereditary hemorrhagictelangiectasia-2, 600376 601313 Polycystic kidney disease, adult type I,173900 601318 Diabetes mellitus, insulin-dependent, 13 601385 Prostatecancer 601412 Deafness, autosomal dominant 7 601471 Moebius syndrome-2601623 Angelman syndrome 601652 Glaucoma 1A, primary open angle,juvenile-onset, 137750 601669 Hirschsprung disease, one form 601682Glaucoma 1C, primary open angle 601744 Systemic lupus erythematosus,susceptibility to, 1 601769 Osteoporosis, involutional 601769 Rickets,vitamin D-resistant, 277440 601777 Cone dystrophy, progressive 601785Carbohydrate-deficient glycoprotein syndrome, type I, 212065 601800[Hair color, brown] 601844 Pseudohypoaldosteronism type II 601846Muscular dystrophy with rimmed vacuoles 601884 [High bone mass] 601889Lymphoma, diffuse large cell 601954 Muscular dystrophy, limb-girdle,type 2G 601969 Medulloblastoma, 155255 601969 Glioblastoma multiforme,137800 601975 Ectodermal dysplasia/skin fragility syndrome 602025Obesity/hyperinsulinism, susceptibility to 602084 Endometrial carcinoma602092 Deafness, autosomal recessive 18 602116 Glioma 602117Prader-Willi syndrome 602134 Tremor, familial essential, 2 602216Peutz-Jeghers syndrome, 175200 602477 Febrile convulsions, familial, 2602491 Hyperlipidemia, familial combined, 1 602568Homocystinuria-megaloblastic anemia, cbl E type, 236270 602629Dystonia-6, torsion 602685 Mental retardation, severe, with spasticityand tapetoretinal degeneration 602759 Prostate cancer, hereditary, 2,176807Mature Polypeptides

The present invention also encompasses mature forms of a polypeptidehaving the amino acid sequence of SEQ ID NO:Y and/or the amino acidsequence encoded by the cDNA in a deposited clone. Polynucleotidesencoding the mature forms (such as, for example, the polynucleotidesequence in SEQ ID NO:X and/or the polynucleotide sequence contained inthe cDNA of a deposited clone) are also encompassed by the invention.Moreover, fragments or variants of these polypeptides (such as,fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to these polypeptides, orpolypeptides encoded by a polynucleotide that hybridizes under stringentconditions to the complementary strand of the polynucleotide encodingthese polypeptides) are also encompassed by the invention. In preferredembodiments, these fragments or variants retain one or more functionalacitivities of the full-length or mature form of the polypeptide (e.g.,biological activity (such as, for example, activity useful in detecting,preventing, diagnosing, prognosticating, treating, and/or amelioratingdiabetes mellitus), antigenicity (ability to bind, or compete with apolypeptide of the invention for binding, to an anti-polypeptide of theinvention antibody), immunogenicity (ability to generate antibody whichbinds to a specific polypeptide of the invention), ability to formmultimers with polypeptides of the invention, and ability to bind to areceptor or ligand for a polypeptide of the invention). Antibodies thatbind the polypeptides of the invention, and polynucleotides encodingthese polypeptides are also encompassed by the invention.

According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretary leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that cleavage specificity of a secretedprotein is ultimately determined by the primary structure of thecomplete protein, that is, it is inherent in the amino acid sequence ofthe polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:46834690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues —13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secretedpolypeptide was analyzed by a computer program called SignalP (HenrikNielsen et al., Protein Engineering 10:1-6 (1997)), which predicts thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis of the amino acidsequences of the secreted proteins described herein by this programprovided the results shown in Table 1A.

In specific embodiments, polypeptides of the invention comprise, oralternatively consist of, the predicted mature form of the polypeptideas delineated in columns 14 and 15 of Table 1A. Moreover, fragments orvariants of these polypeptides (such as, fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to these polypeptides, or polypeptides encoded by apolynucleotide that hybridizes under stringent conditions to thecomplementary strand of the polynucleotide encoding these polypeptides)are also encompassed by the invention. In preferred embodiments, thesefragments or variants retain one or more functional acitivities of thefull-length or mature form of the polypeptide (e.g., biological activity(such as, for example, activity useful in detecting, preventing,diagnosing, prognosticating, treating, and/or ameliorating diabetesmellitus), antigenicity (ability to bind, or compete with a polypeptideof the invention for binding, to an anti-polypeptide of the inventionantibody), immunogenicity (ability to generate antibody which binds to aspecific polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide of the invention). Antibodies that bind thepolypeptides of the invention, and polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Polynucleotides encoding proteins comprising, or consisting of, thepredicted mature form of polypeptides of the invention (e.g.,polynucleotides having the sequence of SEQ ID NO: X (Table 1A, column4), the sequence delineated in columns 7 and 8 of Table 1A, and asequence encoding the mature polypeptide delineated in columns 14 and 15of Table 1A (e.g., the sequence of SEQ ID NO:X encoding the maturepolypeptide delineated in columns 14 and 15 of Table 1)) are alsoencompassed by the invention, as are fragments or variants of thesepolynucleotides (such as, fragments as described herein, polynucleotidesat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothese polyncueotides, and nucleic acids which hybridizes under stringentconditions to the complementary strand of the polynucleotide).

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the present invention provides secretedpolypeptides having a sequence shown in SEQ ID NO:Y which have anN-terminus beginning within 15 residues of the predicted cleavage point(i.e., having 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, or 15 moreor less contiguous residues of SEQ ID NO:Y at the N-termrinus whencompared to the predicted mature form of the polypeptide (e.g., themature polypeptide delineated in columns 14 and 15 of Table 1).Similarly, it is also recognized that in some cases, cleavage of thesignal sequence from a secreted protein is not entirely uniform,resulting in more than one secreted species. These polypeptides, and thepolynucleotides encoding such polypeptides, are contemplated by thepresent invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as desribedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is also directed to variants of the polynucleotidesequence disclosed in SEQ ID NO:X or the complementary strand thereto,nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, thenucleotide sequence of SEQ ID NO:X that encodes the polypeptide sequenceas defined in columns 13 and 14 of Table 1A, nucleotide sequencesencoding the polypeptide sequence as defined in columns 13 and 14 ofTable 1A, the nucleotide sequence of SEQ ID NO:X encoding thepolypeptide sequence as defined in Table 1B, nucleotide sequencesencoding the polypeptide as defined in Table 1B, the nucleotide sequenceas defined in columns 8 and 9 of Table 2, nucleotide sequences encodingthe polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2, the nucleotide sequence as defined in column 6 ofTable 1C, nucleotide sequences encoding the polypeptide encoded by thenucleotide sequence as defined in column 6 of Table 1C, the cDNAsequence contained in ATCC Deposit No:Z, nucleotide sequences encodingthe polypeptide encoded by the cDNA sequence contained in ATCC DepositNo:Z, and/or nucleotide sequences encoding a mature (secreted)polypeptide encoded by the cDNA sequence contained in ATCC Deposit No:Z.

The present invention also encompasses variants of the polypeptidesequence disclosed in SEQ ID NO:Y, the polypeptide as defined in columns13 and 14 of Table 1A, the polypeptide sequence as defined in Table 1B,a polypeptide sequence encoded by the polynucleotide sequence in SEQ IDNO:X, a polypeptide sequence encoded by the nucleotide sequence asdefined in columns 8 and 9 of Table 2, a polypeptide sequence encoded bythe nucleotide sequence as defined in column 6 of Table 1C, apolypeptide sequence encoded by the complement of the polynucleotidesequence in SEQ ID NO:X, the polypeptide sequence encoded by the cDNAsequence contained in ATCC Deposit No:Z and/or a mature (secreted)polypeptide encoded by the cDNA sequence contained in ATCC Deposit No:Z.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence described in SEQ ID NO:X or contained in the cDNAsequence of ATCC Deposit No:Z; (b) a nucleotide sequence in SEQ ID NO:Xor the cDNA in ATCC Deposit No:Z which encodes the complete amino acidsequence of SEQ ID NO:Y or the complete amino acid sequence encoded bythe cDNA in ATCC Deposit No:Z; (c) a nucleotide sequence in SEQ ID NO:Xor the cDNA in ATCC Deposit No:Z which encodes a mature polypeptide(i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15of Table 1A)); (d) a nucleotide sequence in SEQ ID NO:X or the cDNAsequence of ATCC Deposit No:Z, which encodes a biologically activefragment of a polypeptide; (e) a nucleotide sequence in SEQ ID NO:X orthe cDNA sequence of ATCC Deposit No:Z, which encodes an antigenicfragment of a polypeptide; (f) a nucleotide sequence encoding apolypeptide comprising the complete amino acid sequence of SEQ ID NO:Yor the complete amino acid sequence encoded by the cDNA in ATCC DepositNo:Z; (g) a nucleotide sequence encoding a mature polypeptide of theamino acid sequence of SEQ ID NO:Y (i.e., a secreted polypeptide (e.g.,as delineated in columns 14 and 15 of Table 1A)) or a mature polypeptideof the amino acid sequence encoded by the cDNA in ATCC Deposit No:Z; (h)a nucleotide sequence encoding a biologically active fragment of apolypeptide having the complete amino acid sequence of SEQ ID NO:Y orthe complete amino acid sequence encoded by the cDNA in ATCC DepositNo:Z; (i) a nucleotide sequence encoding an antigenic fragment of apolypeptide having the complete amino acid sequence of SEQ ID NO:Y orthe complete amino acid sequence encoded by the cDNA in ATCC DepositNo:Z; and (j) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i)above.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, forexample, any of the nucleotide sequences in (a), (b), (c), (d), (e),(f), (g), (h), (i), or () above, the nucleotide coding sequence in SEQID NO:X or the complementary strand thereto, the nucleotide codingsequence of the cDNA contained in ATCC Deposit No:Z or the complementarystrand thereto, a nucleotide sequence encoding the polypeptide of SEQ IDNO:Y, a nucleotide sequence encoding a polypeptide sequence encoded bythe nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encodedby the complement of the polynucleotide sequence in SEQ ID NO:X, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in ATCC Deposit No:Z, the nucleotide coding sequence in SEQ IDNO:X as defined in columns 8 and 9 of Table 2 or the complementarystrand thereto, a nucleotide sequence encoding the polypeptide encodedby the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9of Table 2 or the complementary strand thereto, the nucleotide codingsequence in SEQ ID NO:B as defined in column 6 of Table 1C or thecomplementary strand thereto, a nucleotide sequence encoding thepolypeptide encoded by the nucleotide sequence in SEQ ID NO:B as definedin column 6 of Table 1C or the complementary strand thereto, thenucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence asdefined in Table 1B or the complementary strand thereto, nucleotidesequences encoding the polypeptide as defined in Table 1B or thecomplementary strand thereto, and/or polynucleotide fragments of any ofthese nucleic acid molecules (e.g., those fragments described herein).Polynucleotides which hybridize to the complement of these nucleic acidmolecules under stringent hybridization conditions or alternatively,under lower stringency conditions, are also encompassed by theinvention, as are polypeptides encoded by these polynucleotides andnucleic acids.

In a preferred embodiment, the invention encompasses nucleic acidmolecules which comprise, or alternatively, consist of a polynucleotidewhich hybridizes under stringent hybridization conditions, oralternatively, under lower stringency conditions, to a polynucleotide in(a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as arepolypeptides encoded by these polynucleotides. In another preferredembodiment, polynucleotides which hybridize to the complement of thesenucleic acid molecules under stringent hybridization conditions, oralternatively, under lower stringency conditions, are also encompassedby the invention, as are polypeptides encoded by these polynucleotides.

In another embodiment, the invention provides a purified proteincomprising, or alternatively consisting of, a polypeptide having anamino acid sequence selected from the group consisting of: (a) thecomplete amino acid sequence of SEQ ID NO:Y or the complete amino acidsequence encoded by the cDNA in ATCC Deposit No:Z; (b) the amino acidsequence of a mature (secreted) form of a polypeptide having the aminoacid sequence of SEQ ID NO:Y (e.g., as delineated in columns 14 and 15of Table 1A) or a mature form of the amino acid sequence encoded by thecDNA in ATCC Deposit No:Z mature; (c) the amino acid sequence of abiologically active fragment of a polypeptide having the complete aminoacid sequence of SEQ ID NO:Y or the complete amino acid sequence encodedby the cDNA in ATCC Deposit No:Z; and (d) the amino acid sequence of anantigenic fragment of a polypeptide having the complete amino acidsequence of SEQ ID NO:Y or the complete amino acid sequence encoded bythe cDNA in ATCC Deposit No:Z.

The present invention is also directed to proteins which comprise, oralternatively consist of, an amino acid sequence which is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example,any of the amino acid sequences in (a), (b), (c), or (d), above, theamino acid sequence shown in SEQ ID NO:Y, the amino acid sequenceencoded by the cDNA contained in ATCC Deposit No:Z, the amino acidsequence of the polypeptide encoded by the nucleotide sequence in SEQ IDNO:X as defined in columns 8 and 9 of Table 2, the amino acid sequenceof the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B asdefined in column 6 of Table 1C, the amino acid sequence as defined inTable 1B, an amino acid sequence encoded by the nucleotide sequence inSEQ ID NO:X, and an amino acid sequence encoded by the complement of thepolynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptidesare also provided (e.g., those fragments described herein). Furtherproteins encoded by polynucleotides which hybridize to the complement ofthe nucleic acid molecules encoding these amino acid sequences understringent hybridization conditions or alternatively, under lowerstringency conditions, are also encompassed by the invention, as are thepolynucleotides encoding these proteins.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence referred to inTable 1B or 2 as the ORF (open reading frame), or any fragment specifiedas described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the present invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is expressed as percent identity. Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence of a polypeptide referred to in Table 1A (e.g., theamino acid sequence delineated in columns 14 and 15) or a fragmentthereof, Table 1B.1 (e.g., the amino acid sequence identified in column6) or a fragment thereof, Table 2 (e.g., the amino acid sequence of thepolypeptide encoded by the polynucleotide sequence defined in columns 8and 9 of Table 2) or a fragment thereof, the amino acid sequence of thepolypeptide encoded by the polynucleotide sequence in SEQ D NO:B asdefined in column 6 of Table 1C or a fragment thereof, the amino acidsequence of the polypeptide encoded by the nucleotide sequence in SEQ IDNO:X or a fragment thereof, or the amino acid sequence of thepolypeptide encoded by cDNA contained in ATCC Deposit No:Z, or afragment thereof, the amino acid sequence of a mature (secreted)polypeptide encoded by cDNA contained in ATCC Deposit No:Z, or afragment thereof, can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is expressed as percent identity. Preferredparameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5,Gap Size Penalty=0.05, Window Size500 or the length of the subject aminoacid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N— orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N— and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N— and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N— and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N— and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N— andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N— and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N— orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N—and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequnce aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The polynucleotide variants of the invention may contain alterations inthe coding regions, non-coding regions, or both. Especially preferredare polynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, polypeptide variants in which less than 50, lessthan 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10,1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination are also preferred. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the polypeptide of the present invention withoutsubstantial loss of biological function. As an example, Ron et al. (J.Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins havingheparin binding activity even after deleting 3, 8, or 27 amino-terminalamino acid residues. Similarly, Interferon gamma exhibited up to tentimes higher activity after deleting 8-10 amino acid residues from thecarboxy terminus of this protein. (Dobeli et al., J. Biotechnology7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N— or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show abiological or functional activity of the polypeptides of the invention(such as, for example, activity useful in detecting, preventing,diagnosing, prognosticating, treating, and/or ameliorating diabetesmellitus). Such variants include deletions, insertions, inversions,repeats, and substitutions selected according to general rules known inthe art so as have little effect on activity.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleicacid sequences disclosed herein, (e.g., encoding a polypeptide havingthe amino acid sequence of an N and/or C terminal deletion),irrespective of whether they encode a polypeptide having functionalactivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having functional activity, one of skillin the art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having functional activity include, interalia, (1) isolating a gene or allelic or splice variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of the gene,as described in Verma et al., Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York (1988); (3) Northern Blot analysisfor detecting mRNA expression in specific tissues (e.g., normal ordiseased tissues); and (4) in situ hybridization (e.g., histochemistry)for detecting mRNA expression in specific tissues (e.g., normal ordiseased tissues).

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleicacid sequences disclosed herein, which do, in fact, encode a polypeptidehaving functional activity. By a polypeptide having “functionalactivity” is meant, a polypeptide capable of displaying one or moreknown functional activities associated with a full-length (complete)protein and/or a mature (secreted) protein of the invention. Suchfunctional activities include, but are not limited to, biologicalactivity (such as, for example, activity useful in detecting,preventing, diagnosing, prognosticating, treating, and/or amelioratingdiabetes mellitus), antigenicity (ability to bind, or compete with apolypeptide of the invention for binding, to an anti-polypeptide of theinvention antibody), immunogenicity (ability to generate antibody whichbinds to a specific polypeptide of the invention), ability to formmultimers with polypeptides of the invention, and ability to bind to areceptor or ligand for a polypeptide of the invention.

The functional activity of the polypeptides, and fragments, variants andderivatives of the invention, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with a full-length polypeptide of the present inventionfor binding to an anti-polypetide antibody, various immunoassays knownin the art can be used, including but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a ligand is identified, or the ability of apolypeptide fragment, variant or derivative of the invention tomultimerize is being evaluated, binding can be assayed, e.g., by meanswell-known in the art, such as, for example, reducing and non-reducinggel chromatography, protein affinity chromatography, and affinityblotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123(1995). In another embodiment, the ability of physiological correlatesof a polypeptide of the present invention to bind to a substrate(s) ofthe polypeptide of the invention can be routinely assayed usingtechniques known in the art.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability ofpolypeptides of the present invention and fragments, variants andderivatives thereof to elicit polypeptide related biological activity(either in vitro or in vivo). Other methods will be known to the skilledartisan and are within the scope of the invention.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acidsequence of the cDNA contained in ATCC Deposit No:Z, the nucleic acidsequence referred to in Table 1B (SEQ ID NO:X), the nucleic acidsequence disclosed in Table 1A (e.g., the nucleic acid sequencedelineated in columns 7 and 8), the nucleic acid sequence disclosed inTable 2 (e.g., the nucleic acid sequence delineated in columns 8 and 9)or fragments thereof, will encode polypeptides “having functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having functionalactivity. This is because the skilled artisan is fully aware of aminoacid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. See Cunningham and Wells,Science 244:1081-1085 (1989). The resulting mutant molecules can then betested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of the presentinvention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitutions with one or more of the amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), (iv)fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, serum albumin (preferablyhuman serum albumin) or a fragment thereof, or leader or secretorysequence, or a sequence facilitating purification, or (v) fusion of thepolypeptide with another compound, such as albumin (including but notlimited to recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). Such variant polypeptides are deemed to be within the scopeof those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

A further embodiment of the invention relates to polypeptides whichcomprise the amino acid sequence of a polypeptide having an amino acidsequence which contains at least one amino acid substitution, but notmore than 50 amino acid substitutions, even more preferably, not morethan 40 amino acid substitutions, still more preferably, not more than30 amino acid substitutions, and still even more preferably, not morethan 20 amino acid substitutions from a polypeptide sequence disclosedherein. Of course it is highly preferable for a polypeptide to have anamino acid sequence which, for example, comprises the amino acidsequence of a polypeptide of SEQ ID NO:Y, the amino acid sequence of themature (e.g., secreted) polypeptide of SEQ ID NO:Y, an amino acidsequence encoded by SEQ ID NO:X, an amino acid sequence encoded by theportion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, anamino acid sequence encoded by the complement of SEQ ID NO:X, an aminoacid sequence encoded by cDNA contained in ATCC Deposit No:Z, and/or theamino acid sequence of a mature (secreted) polypeptide encoded by cDNAcontained in ATCC Deposit No:Z, or a fragment thereof, which contains,in order of ever-increasing preference, at least one, but not more than10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.

In specific embodiments, the polypeptides of the invention comprise, oralternatively, consist of, fragments or variants of a reference aminoacid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Yor fragments thereof (e.g., the mature form and/or other fragmentsdescribed herein); (b) the amino acid sequence encoded by SEQ ID NO:X orfragments thereof; (c) the amino acid sequence encoded by the complementof SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encodedby the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2or fragments thereof; and (e) the amino acid sequence encoded by cDNAcontained in ATCC Deposit No:Z or fragments thereof; wherein thefragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, aminoacid residue additions, substitutions, and/or deletions when compared tothe reference amino acid sequence. In preferred embodiments, the aminoacid substitutions are conservative. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments ofthe polynucleotides (nucleic acids) of the invention. In the presentinvention, a “polynucleotide fragment” refers to a polynucleotide havinga nucleic acid sequence which, for example: is a portion of the cDNAcontained in ATCC Deposit No:Z or the complementary strand thereto; is aportion of the polynucleotide sequence encoding the polypeptide encodedby the cDNA contained in ATCC Deposit No:Z or the complementary strandthereto; is a portion of the polynucleotide sequence encoding the mature(secreted) polypeptide encoded by the cDNA contained in ATCC DepositNo:Z or the complementary strand thereto; is a portion of apolynucleotide sequence encoding the mature amino acid sequence asdefined in columns 14 and 15 of Table 1A or the complementary strandthereto; is a portion of a polynucleotide sequence encoding the aminoacid sequence encoded by the region of SEQ ID NO:X as defined in columns8 and 9 of Table 2 or the complementary strand thereto; is a portion ofthe polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9of Table 2 or the complementary strand thereto; is a portion of thepolynucleotide sequence in SEQ ID NO:X or the complementary strandthereto; is a polynucleotide sequence encoding a portion of thepolypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding aportion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotidesequence encoding a portion of a polypeptide encoded by the complementof the polynucleotide sequence in SEQ ID NO:X; is a portion of apolynucleotide sequence encoding the amino acid sequence encoded by theregion of SEQ ID NO:B as defined in column 6 of Table 1C or thecomplementary strand thereto; or is a portion of the polynucleotidesequence of SEQ ID NO:B as defined in column 6 of Table 1C or thecomplementary strand thereto.

The polynucleotide fragments of the invention are preferably at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt, at least about 50 nt, at least about 75 nt, or at leastabout 150 nt in length. A fragment “at least 20 nt in length,” forexample, is intended to include 20 or more contiguous bases from thecDNA sequence contained in ATCC Deposit No:Z, or the nucleotide sequenceshown in SEQ ID NO:X or the complementary stand thereto. In this context“about” includes the particularly recited value or a value larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. These nucleotide fragments have uses that include, butare not limited to, as diagnostic probes and primers as discussedherein. Of course, larger fragments (e.g., at least 160, 170, 180, 190,200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are alsoencompassed by the invention.

Moreover, representative examples of polynucleotide fragments of theinvention comprise, or alternatively consist of, a sequence from aboutnucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,301-350, 351400, 401450, 451-500, 501-550, 551-600, 601-650, 651-700,701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050,1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250,2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550,2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850,2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150,3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450,3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750,3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050,4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350,4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650,4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950,4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250,5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550,5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850,5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150,6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450,6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750,6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050,7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the endof SEQ ID NO:X, or the complementary strand thereto. In this context“about” includes the particularly recited range or a range larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. Preferably, these fragments encode a polypeptide whichhas a functional activity (e.g., biological activity; such as, forexample, activity useful in detecting, preventing, diagnosing,prognosticating, treating, and/or ameliorating diabetes mellitus). Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Polynucleotides which hybridize to one or more ofthese polynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions are also encompassed bythe invention, as are polypeptides encoded by these polynucleotides.

Further representative examples of polynucleotide fragments of theinvention comprise, or alternatively consist of, a sequence from aboutnucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,301-350, 351400, 401450, 451-500, 501-550, 551-600, 601-650, 651-700,701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050,1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250,2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550,2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850,2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150,3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450,3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750,3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050,4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350,4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650,4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950,4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250,5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550,5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850,5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150,6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450,6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750,6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050,7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the endof the cDNA sequence contained in ATCC Deposit No:Z, or thecomplementary strand thereto. In this context “about” includes theparticularly recited range or a range larger or smaller by several (5,4, 3, 2, or 1) nucleotides, at either terminus or at both termini.Preferably, these fragments encode a polypeptide which has a functionalactivity (e.g., biological activity). More preferably, thesepolynucleotides can be used as probes or primers as discussed herein.Polynucleotides which hybridize to one or more of these polynucleotidesunder stringent hybridization conditions or alternatively, under lowerstringency conditions are also encompassed by the invention, as arepolypeptides encoded by these polynucleotides.

Moreover, representative examples of polynucleotide fragments of theinvention comprise, or alternatively consist of, a nucleic acid sequencecomprising one, two, three, four, five, six, seven, eight, nine, ten, ormore of the above described polynucleotide fragments of the invention incombination with a polynucleotide sequence delineated in Table 1C column6. Additional, representative examples of polynucleotide fragments ofthe invention comprise, or alternatively consist of, a nucleic acidsequence comprising one, two, three, four, five, six, seven, eight,nine, ten, or more of the above described polynucleotide fragments ofthe invention in combination with a polynucleotide sequence that is thecomplementary strand of a sequence delineated in column 6 of Table 1C.In further embodiments, the above-described polynucleotide fragments ofthe invention comprise, or alternatively consist of, sequencesdelineated in Table 1C, column 6, and have a nucleic acid sequence whichis different from that of the BAC fragment having the sequence disclosedin SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, theabove-described polynucleotide fragments of the invention comprise, oralternatively consist of, sequences delineated in Table 1C, column 6,and have a nucleic acid sequence which is different from that publishedfor the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Inadditional embodiments, the above-described polynucleotides of theinvention comprise, or alternatively consist of, sequences delineatedTable 1C, column 6, and have a nucleic acid sequence which is differentfrom that contained in the BAC clone identified as BAC ID NO:A (seeTable 1C, column 4). Polypeptides encoded by these polynucleotides,other polynucleotides that encode these polypeptides, and antibodiesthat bind these polypeptides are also encompassed by the invention.Additionally, fragments and variants of the above-describedpolynucleotides and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more fragments of the sequences delineatedin column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X(e.g., as defined in Table 1C, column 2) or fragments or variantsthereof. Polypeptides encoded by these polynucleotides, otherpolynucleotides that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more fragments of the sequences delineatedin column 6 of Table 1C which correspond to the same ATCC Deposit No:Z(see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X(e.g., as defined in Table 1A, 1B, or 1C) or fragments or variantsthereof. Polypeptides encoded by these polynucleotides, otherpolynucleotides that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention.

In further specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of, one, two, three, four, five, six,seven, eight, nine, ten, or more fragments of the sequences delineatedin the same row of column 6 of Table 1C, and the polynucleotide sequenceof SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments orvariants thereof. Polypeptides encoded by these polynucleotides, otherpolynucleotides that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of a polynucleotide sequence in whichthe 3′ 10 polynucleotides of one of the sequences delineated in column 6of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:Xare directly contiguous. Nucleic acids which hybridize to the complementof these 20 contiguous polynucleotides under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention. Polypeptides encoded by thesepolynucleotides and/or nucleic acids, other polynucleotides and/ornucleic acids that encode these polypeptides, and antibodies that bindthese polypeptides are also encompassed by the invention. Additionally,fragments and variants of the above-described polynucleotides, nucleicacids, and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of a polynucleotide sequence in whichthe 3′ 10 polynucleotides of one of the sequences delineated in column 6of Table 1C and the 5′ 10 polynucleotides of a fragment or variant ofthe sequence of SEQ ID NO:X (e.g., as described herein) are directlycontiguous Nucleic acids which hybridize to the complement of these 20contiguous polynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention. Polypeptides encoded by these polynucleotides and/ornucleic acids, other polynucleotides and/or nucleic acids encoding thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides arealso encompassed by the invention.

In further specific embodiments, polynucleotides of the inventioncomprise, or alternatively consist of a polynucleotide sequence in whichthe 3′ 10 polynucleotides of a fragment or variant of the sequence ofSEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of thesequences delineated in column 6 of Table 1C are directly contiguous.Nucleic acids which hybridize to the complement of these 20 contiguouspolynucleotides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassedby the invention. Polypeptides encoded by these polynucleotides and/ornucleic acids, other polynucleotides and/or nucleic acids encoding thesepolypeptides, and antibodies that bind these polypeptides are alsoencompassed by the invention. Additionally, fragments and variants ofthe above-described polynucleotides, nucleic acids, and polypeptides arealso encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, oralternatively consist of a polynucleotide sequence in which the 3′ 10polynucleotides of one of the sequences delineated in column 6 of Table1C and the 5′ 10 polynucleotides of another sequence in column 6 aredirectly contiguous. In preferred embodiments, the 3′ 10 polynucleotidesof one of the sequences delineated in column 6 of Table 1C is directlycontiguous with the 5′ 10 polynucleotides of the next sequential exondelineated in Table 1C, column 6. Nucleic acids which hybridize to thecomplement of these 20 contiguous polynucleotides under stringenthybridization conditions or alternatively, under lower stringencyconditions, are also encompassed by the invention. Polypeptides encodedby these polynucleotides and/or nucleic acids, other polynucleotidesand/or nucleic acids encoding these polypeptides, and antibodies thatbind these polypeptides are also encompassed by the invention.Additionally, fragments and variants of the above-describedpolynucleotides, nucleic acids, and polypeptides are also encompassed bythe invention.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of the amino acid sequence contained inSEQ ID NO:Y, is a portion of the mature form of SEQ ID NO:Y as definedin columns 14 and 15 of Table 1A, a portion of an amino acid sequenceencoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 ofTable 2, is a portion of an amino acid sequence encoded by thepolynucleotide sequence of SEQ ID NO:X, is a portion of an amino acidsequence encoded by the complement of the polynucleotide sequence in SEQID NO:X, is a portion of the amino acid sequence of a mature (secreted)polypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/oris a portion of an amino acid sequence encoded by the cDNA contained inATCC Deposit No:Z. Protein (polypeptide) fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, from about amino acid number 1-20, 2140, 41-60, 61-80,81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240,241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400,401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560,561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720,721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880,881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020,1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140,1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260,1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380,1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding regionof cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptidefragments of the invention include, for example, fragments comprising,or alternatively consisting of, from about amino acid number 1-20,21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340,341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500,501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660,661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820,821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980,981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100,1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220,1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340,1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to theend of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragmentsof the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, or ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities; such as, for example, activity useful in detecting,preventing, diagnosing, prognosticating, treating, and/or amelioratingdiabetes mellitus; ability to multimerize; ability to bind a ligand;antigenic ability useful for production of polypeptide specificantibodies) may still be retained. For example, the ability of shortenedmuteins to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata mutein with a large number of deleted N-terminal amino acid residuesmay retain some biological or immunogenic activities. In fact, peptidescomposed of as few as six amino acid residues may often evoke an immuneresponse.

Accordingly, polypeptide fragments include the secreted protein as wellas the mature form. Further preferred polypeptide fragments include thesecreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted polypeptide or the matureform. Similarly, any number of amino acids, ranging from 1-30, can bedeleted from the carboxy terminus of the secreted protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

The present invention further provides polypeptides having one or moreresidues deleted from the amino terminus of the amino acid sequence of apolypeptide disclosed herein (e.g., a polypeptide of SEQ D NO:Y, apolypeptide as defined in columns 14 and 15 of Table 1A, a polypeptideencoded by the polynucleotide sequence contained in SEQ D NO:X or thecomplement thereof, a polypeptide encoded by the portion of SEQ ID NO:Xas defined in columns 8 and 9 of Table 2, a polypeptide encoded by theportion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptideencoded by the cDNA contained in ATCC Deposit No:Z, and/or a maturepolypeptide encoded by the cDNA contained in ATCC Deposit No:Z). Inparticular, N-terminal deletions may be described by the general formulam-q, where q is a whole integer representing the total number of aminoacid residues in a polypeptide of the invention (e.g., the polypeptidedisclosed in SEQ ED NO:Y, the mature (secreted) portion of SEQ ID NO:Yas defined in columns 14 and 15 of Table 1A, or the polypeptide encodedby the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2),and m is defined as any integer ranging from 2 to q-6. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

The present invention further provides polypeptides having one or moreresidues from the carboxy terminus of the amino acid sequence of apolypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, themature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15of Table 1A, a polypeptide encoded by the polynucleotide sequencecontained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ IDNO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded bythe portion of SEQ ID NO:B as defined in column 6 of Table 1C, apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/or amature polypeptide encoded by the cDNA contained in ATCC Deposit No:Z).In particular, C-terminal deletions may be described by the generalformula 1-n, where n is any whole integer ranging from 6 to q-1, andwhere n corresponds to the position of amino acid residue in apolypeptide of the invention. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

In addition, any of the above described N— or C-terminal deletions canbe combined to produce a N— and C-terminal deleted polypeptide. Theinvention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues m-n of a polypeptide encoded bySEQ ID NO:X (e.g., including, but not limited to, the preferredpolypeptide disclosed as SEQ ID NO:Y, the mature (secreted) portion ofSEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, and thepolypeptide encoded by the portion of SEQ ID NO:X as defined in columns8 and 9 of Table 2), the cDNA contained in ATCC Deposit No:Z, and/or thecomplement thereof, where n and m are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities such as, for example, activity useful indetecting, preventing, diagnosing, prognosticating, treating, and/orameliorating diabetes mellitus; ability to multimerize; ability to binda ligand; antigenic ability useful for production of polypeptidespecific antibodies) may still be retained. For example the ability ofthe shortened mutein to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a mutein with a large number of deleted C-terminal aminoacid residues may retain some biological or immunogenic activities. Infact, peptides composed of as few as six amino acid residues may oftenevoke an immune response.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identicalto a polypeptide sequence set forth herein. In preferred embodiments,the application is directed to proteins containing polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptideshaving the amino acid sequence of the specific N— and C-terminaldeletions. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Any polypeptide sequence encoded by, for example, the polynucleotidesequences set forth as SEQ ID NO:X or the complement thereof,(presented, for example, in Tables 1A and 2), the cDNA contained in ATCCDeposit No:Z, or the polynucleotide sequence as defined in column 6 ofTable 1C, may be analyzed to determine certain preferred regions of thepolypeptide. For example, the amino acid sequence of a polypeptideencoded by a polynucleotide sequence of SEQ ID NO:X (e.g., thepolypeptide of SEQ ID NO:Y and the polypeptide encoded by the portion ofSEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the cDNAcontained in ATCC Deposit No:Z may be analyzed using the defaultparameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S.Park St., Madison, Wis. 53715 USA; http:Hlwww.dnastar.com/).

Polypeptide regions that may be routinely obtained using the DNASTARcomputer algorithm include, but are not limited to, Garnier-Robsonalpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasmanalpha-regions, beta-regions, and turn-regions; Kyte-Doolittlehydrophilic regions and hydrophobic regions; Eisenberg alpha- andbeta-amphipathic regions; Karplus-Schulz flexible regions; Eminisurface-forming regions; and Jameson-Wolf regions of high antigenicindex. Among highly preferred polynucleotides of the invention in thisregard are those that encode polypeptides comprising regions thatcombine several structural features, such as several (e.g., 1, 2, 3 or4) of the features set out above.

Additionally, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Emini surface-forming regions, and Jameson-Wolf regions of highantigenic index (i.e., containing four or more contiguous amino acidshaving an antigenic index of greater than or equal to 1.5, as identifiedusing the default parameters of the Jameson-Wolf program) can routinelybe used to determine polypeptide regions that exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom data by DNASTAR analysis by choosing values which represent regionsof the polypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

Preferred polypeptide fragments of the invention are fragmentscomprising, or alternatively, consisting of, an amino acid sequence thatdisplays a functional activity (e.g. biological activity such as, forexample, activity useful in detecting, preventing, diagnosing,prognosticating, treating, and/or ameliorating diabetes mellitus;ability to multimerize; ability to bind a ligand; antigenic abilityuseful for production of polypeptide specific antibodies) of thepolypeptide sequence of which the amino acid sequence is a fragment. Bya polypeptide displaying a “functional activity” is meant a polypeptidecapable of one or more known functional activities associated with afull-length protein, such as, for example, biological activity,antigenicity, immunogenicity, and/or multimerization, as describedherein.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.

In preferred embodiments, polypeptides of the invention comprise, oralternatively consist of, one, two, three, four, five or more of theantigenic fragments of the polypeptide of SEQ ID NO:Y, or portionsthereof. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of: the polypeptide sequenceshown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X orthe complementary strand thereto; the polypeptide sequence encoded bythe portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; thepolypeptide sequence encoded by the portion of SEQ ID NO:B as defined incolumn 6 of Table 1C or the complement thereto; the polypeptide sequenceencoded by the cDNA contained in ATCC Deposit No:Z; or the polypeptidesequence encoded by a polynucleotide that hybridizes to the sequence ofSEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, thecomplement of a portion of SEQ ID NO:X as defined in columns 8 and 9 ofTable 2, or the cDNA sequence contained in ATCC Deposit No:Z understringent hybridization conditions or alternatively, under lowerstringency hybridization as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:X, or a fragment thereof),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or alternatively, under lower stringencyhybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Imununospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Non-limiting examples of epitopes of polypeptides that can be used togenerate antibodies of the invention include a polypeptide comprising,or alternatively consisting of, at least one, two, three, four, five,six or more of the portion(s) of SEQ ID NO:Y specified in Table 1B.These polypeptide fragments have been determined to bear antigenicepitopes of the proteins of the invention by the analysis of theJameson-Wolf antigenic index which is included in the DNAStar suite ofcomputer programs. By “comprise” it is intended that a polypeptidecontains at least one, two, three, four, five, six or more of theportion(s) of SEQ ID NO:Y shown in Table 1B, but it may containadditional flanking residues on either the amino or carboxyl termini ofthe recited portion. Such additional flanking sequences are preferablysequences naturally found adjacent to the portion; i.e., contiguoussequence shown in SEQ ID NO:Y. The flanking sequence may, however, besequences from a heterolgous polypeptide, such as from another proteindescribed herein or from a heterologous polypeptide not describedherein. In particular embodiments, epitope portions of a polypeptide ofthe invention comprise one, two, three, or more of the portions of SEQID NO:Y shown in Table 1B.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acids1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N— or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (HA) tag or flag tag) to aid in detection and purificationof the expressed polypeptide. For example, a system described byJanknecht et al. allows for the ready purification of non-denaturedfusion proteins expressed in human cell lines (Janknecht et al., 1991,Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene ofinterest is subcloned into a vaccinia recombination plasmid such thatthe open reading frame of the gene is translationally fused to anamino-terminal tag consisting of six histidine residues. The tag servesas a matrix binding domain for the fusion protein. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because secreted proteins target cellular locations based ontrafficking signals, polypeptides of the present invention which areshown to be secreted can be used as targeting molecules once fused toother proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

In certain preferred embodiments, proteins of the invention are fusionproteins comprising an amino acid sequence that is an N and/orC-terminal deletion of a polypeptide of the invention. In preferredembodiments, the invention is directed to a fusion protein comprising anamino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99%identical to a polypeptide sequence of the invention. Polynucleotidesencoding these proteins are also encompassed by the invention.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

As one of skill in the art will appreciate that, as discussed above,polypeptides of the present invention, and epitope-bearing fragmentsthereof, can be combined with heterologous polypeptide sequences. Forexample, the polypeptides of the present invention may be fused withheterologous polypeptide sequences, for example, the polypeptides of thepresent invention may be fused with the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3,and any combination thereof, including both entire domains and portionsthereof), or albumin (including, but not limited to, native orrecombinant human albumin or fragments or variants thereof (see, e.g.,U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, andU.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated byreference in their entirety)), resulting in chimeric polypeptides. Forexample, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusionproteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties (EP-A 0232 262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a polypeptide which facilitates purificationof the fused polypeptide. In preferred embodiments, the marker aminoacid sequence is a hexa-histidine peptide, such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)).

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:X and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Recombinant and Synthetic Production of Polypeptides of the Invention

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by synthetic and recombinant techniques. The vector maybe, for example, a phage, plasmid, viral, or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides of the invention may be joined to a vectorcontaining a selectable marker for propagation in a host. Generally, aplasmid vector is introduced in a precipitate, such as a calciumphosphate precipitate, or in a complex with a charged lipid. If thevector is a virus, it may be packaged in vitro using an appropriatepackaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418, glutamine synthase, or neomycin resistance for eukaryotic cellculture, and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris(ATCC Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated intheir entireties by reference herein. Additionally, glutamine synthaseexpression vectors can be obtained from Lonza Biologics, Inc.(Portsmouth, N.H.). Expression and production of monoclonal antibodiesusing a GS expression system in murine myeloma cells is described inBebbington et al., Bio/technology 10:169(1992) and in Biblia andRobinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated byreference.

The present invention also relates to host cells containing theabove-described vector constructs described herein, and additionallyencompasses host cells containing nucleotide sequences of the inventionthat are operably associated with one or more heterologous controlregions (e.g., promoter and/or enhancer) using techniques known of inthe art. The host cell can be a higher eukaryotic cell, such as amammalian cell (e.g., a human derived cell), or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. A host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of theinvention into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986). It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., the coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with polynucleotides of the invention, and which activates,alters, and/or amplifies endogenous polynucleotides. For example,techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination (see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication Number WO 96/29411; International Publication Number WO94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of eachof which are incorporated by reference in their entireties).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention can also be recovered from:products purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In one embodiment, the yeast Pichia pastoris is used to expresspolypeptides of the invention in a eukaryotic system. Pichia pastoris isa methylotrophic yeast which can metabolize methanol as its sole carbonsource. A main step in the methanol metabolization pathway is theoxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. SeeEllis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a polypeptide of the invention by virtue ofthe strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHEL-D2, pH[L-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with polynucleotides of the invention, and which activates,alters, and/or amplifies endogenous polynucleotides. For example,techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination (see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., New York, andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide can besynthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, omithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,fluoro-amino acids, designer amino acids such as b-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

The invention encompasses polypeptides of the present invention whichare differentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBf₄; acetylation,formylation, oxidation, reduction; metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidinibiotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include iodine (¹²¹I,¹²³I, ¹²⁵I, ¹³¹I, carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium (⁹⁹Tc,^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, 75Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

In specific embodiments, a polypeptide of the present invention orfragment or variant thereof is attached to macrocyclic chelators thatassociate with radiometal ions, including but not limited to, ¹⁷⁷Lu,⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferred embodiment, theradiometal ion associated with the macrocyclic chelators is ¹¹¹In. Inanother preferred embodiment, the radiometal ion associated with themacrocyclic chelator is ⁹⁰Y. In specific embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA). In other specific embodiments, DOTA is attached to an antibodyof the invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); whichare hereby incorporated by reference in their entirety.

As mentioned, the proteins of the invention may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide.Polypeptides of the invention may be branched, for example, as a resultof ubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646(1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycolipropylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, such as, for example, themethod disclosed in EP 0 401 384 (coupling PEG to G-CSF), hereinincorporated by reference; see also Malik et al., Exp. Hematol.20:1028-1035 (1992), reporting pegylation of GM-CSF using tresylchloride. For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin International Publication No. WO 98/32466, the entire disclosure ofwhich is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 46, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention can be recovered and purified fromchemical synthesis and recombinant cell cultures by standard methodswhich include, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and/or purification.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer refers to a multimer containing onlypolypeptides corresponding to a protein of the invention (e.g., theamino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded bySEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequenceencoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 ofTable 2, and/or an amino acid sequence encoded by cDNA contained in ATCCDeposit No:Z (including fragments, variants, splice variants, and fusionproteins, corresponding to these as described herein)). These homomersmay contain polypeptides having identical or different amino acidsequences. In a, specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing two polypeptides having identical ordifferent amino acid sequences) or a homotrimer (e.g., containing threepolypeptides having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked by, for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in SEQ IDNO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and9 of Table 2, and/or encoded by the cDNA contained in ATCC DepositNo:Z). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCr application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, proteins of the invention are associated byinteractions between heterologous polypeptide sequence contained inFlag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of the invention (e.g., a polypeptideor fragment or variant of the amino acid sequence of SEQ ID NO:Y or apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z, and/oran epitope, of the present invention) as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding.Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), intracellularly-made antibodies (i.e., intrabodies), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Inpreferred embodiments, the immunoglobulin molecules of the invention areIgG1. In other preferred embodiments, the immunoglobulin molecules ofthe invention are IgG4.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, or by size in contiguous amino acidresidues, or listed in the Tables and Figures. Preferred epitopes of theinvention include the predicted epitopes shown in Table 1B, as well aspolynucleotides that encode these epitopes. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M,5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 11l(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, to purify,detect, and target the polypeptides of the present invention, includingboth in vitro and in vivo diagnostic and therapeutic methods. Forexample, the antibodies have utility in immunoassays for qualitativelyand quantitatively measuring levels of the polypeptides of the presentinvention in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);incorporated by reference herein in its entirety.

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N— or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387; the disclosures of which are incorporatedherein by reference in their entireties.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples. In a non-limiting example, mice canbe immunized with a polypeptide of the invention or a cell expressingsuch peptide. Once an immune response is detected, e.g., antibodiesspecific for the antigen are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, New York, which is hereby incorporated in its entirety byreference. The source of B cells for transformation is commonly humanperipheral blood, but B cells for transformation may also be derivedfrom other sources including, but not limited to, lymph nodes, tonsil,spleen, tumor tissue, and infected tissues. Tissues are generally madeinto single cell suspensions prior to EBV transformation. Additionally,steps may be taken to either physically remove or inactivate T cells(e.g., by treatment with cyclosporin A) in B cell-containing samples,because T cells from individuals seropositive for anti-EBV antibodiescan suppress B cell immortalization by EBV.

In general, the sample containing human B cells is innoculated with EBV,and cultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand(s)/receptor(s).For example, such anti-idiotypic antibodies can be used to bind apolypeptide of the invention and/or to bind its ligand(s)/receptor(s),and thereby block its biological activity. Alternatively, antibodieswhich bind to and enhance polypeptide multimerization and/or binding,and/or receptor/ligand multimerization, binding and/or signaling can beused to generate anti-idiotypes that function as agonists of apolypeptide of the invention and/or its ligand/receptor. Such agonisticanti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens as agonists of the polypeptides of the invention orits ligand(s)/receptor(s). For example, such anti-idiotypic antibodiescan be used to bind a polypeptide of the invention and/or to bind itsligand(s)/receptor(s), and thereby promote or enhance its biologicalactivity.

Intrabodies of the invention can be produced using methods known in theart, such as those disclosed and reviewed in Chen et al., Hum. GeneTher. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997);Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba etal., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz andSteipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods231:207-222 (1999); and references cited therein.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or alternatively, under lower stringency hybridizationconditions, e.g., as defined supra, to polynucleotides that encode anantibody, preferably, that specifically binds to a polypeptide of theinvention, preferably, an antibody that binds to a polypeptide havingthe amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by aportion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/orto a polypeptide encoded by the cDNA contained in ATCC Deposit No:Z.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, New York, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques. Methodsof producing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCrPublication WO 86/05807; PCI Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non- essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts. (e.g., see Logan& Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147(1984)). Methods commonly known in the art of recombinant DNA technologymay be routinely applied to select the desired recombinant clone, andsuch methods are described, for example, in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, New York(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York (1990); and in Chapters 12 and 13, Dracopoli etal. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NewYork (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), whichare incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/01036; WO89/10404; and WO91/06657 which are incorporated in theirentireties by reference herein. Additionally, glutamine synthaseexpression vectors that may be used according to the present inventionare commercially available from suppliers, including, for example LonzaBiologics, Inc. (Portsmouth, N.H.). Expression and production ofmonoclonal antibodies using a GS expression system in murine myelomacells is described in Bebbington et al., Bio/technology 10:169(1992) andin Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which areincorporated in their entirities by reference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCIpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:Y may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:Y maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964(1995). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. See, for example, EP A 232,262.Alternatively, deleting the Fc part after the fusion protein has beenexpressed, detected, and purified, would be desired. For example, the Fcportion may hinder therapy and diagnosis if the fusion protein is usedas an antigen for immunizations. In drug discovery, for example, humanproteins, such as hIL-5, have been fused with Fc portions for thepurpose of high-throughput screening assays to identify antagonists ofhIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995);Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidintbiotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, B-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamnide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. Translation products of the gene ofthe present invention may be useful as cell-specific markers, or morespecifically as cellular markers that are differentially expressed atvarious stages of differentiation and/or maturation of particular celltypes. Monoclonal antibodies directed against a specific epitope, orcombination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:73749 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, section 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, section 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the arL For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, section 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Antibodies of the invention may be characterized usingimmunocytochemisty methods on cells (e.g., mammalian cells, such as CHOcells) transfected with a vector enabling the expression of an antigenor with vector alone using techniques commonly known in the art.Antibodies that bind antigen transfected cells, but not vector-onlytransfected cells, are antigen specific.

Therapeutic Uses

Table 1D also provides information regarding biological activities andpreferred therapeutic uses (i.e. see, “Preferred Indications” column)for polynucleotides and polypeptides of the invention (includingantibodies, agonists, and/or antagonists thereof). Table 1D alsoprovides information regarding assays which may be used to testpolynucleotides and polypeptides of the invention (including antibodies,agonists, and/or antagonists thereof) for the corresponding biologicalactivities. The first column (“Gene No.”) provides the gene number inthe application for each clone identifier. The second column (“cDNA ATCCDeposit No:Z”) provides the unique clone identifier for each clone aspreviously described and indicated in Table 1A, Table 1B, and Table 1C.The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQID Number for polypeptide sequences encoded by the corresponding cDNAclones (also as indicated in Table 1A, Table 1B, and Table 2). Thefourth column (“Biological Activity”) indicates a biological activitycorresponding to the indicated polypeptides (or polynucleotides encodingsaid polypeptides). The fifth column (“Exemplary Activity Assay”)further describes the corresponding biological activity and alsoprovides information pertaining to the various types of assays which maybe performed to test, demonstrate, or quantify the correspondingbiological activity.

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to detect, prevent,diagnose, prognosticate, treat, and/or ameliorate diseases, disorders orconditions associated with aberrant expression and/or activity of apolypeptide of the invention, including, but not limited to, diabetesmellitus. The treatment and/or prevention of diabetes mellitusassociated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptomsassociated with diabetes mellitus. Antibodies of the invention may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

In a specific and preferred embodiment, the present invention isdirected to antibody-based therapies which involve administeringantibodies of the invention to an animal, preferably a mammal, and mostpreferably a human, patient for treating diabetes mellitus. Therapeuticcompounds of the invention include, but are not limited to, antibodiesof the invention (e.g., antibodies directed to the full length proteinexpressed on the cell surface of a mammalian cell; antibodies directedto an epitope of a polypeptide of the invention (such as, for example, apredicted linear epitope shown in Table 1B; or a conformational epitope,including fragments, analogs and derivatives thereof as describedherein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof and anti-idiotypicantibodies as described herein). The antibodies of the invention can beused to detect, diagnose, prevent, treat, prognosticate, and/orameliorate diabetes mellitus or conditions associated with aberrantexpression and/or activity of a polypeptide of the invention. Thetreatment and/or prevention of diabetes mellitus or conditionsassociated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. Antibodies ofthe invention may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diabetes mellitus related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a diabetes mellitus associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, New York (1993); and Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NewYork (1990).

In a preferred embodiment, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijistra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92106180; WO 92122635;WO92120316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94112649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby the presence or absence of an appropriate inducer of transcription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably a polypeptide orantibody of the invention. In a preferred embodiment, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353- 365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) In yetanother embodiment, the compound or composition can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, e.g., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopoeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, prognosticate, or monitordiabetes mellitus and/or conditions associated with the aberrantexpression and/or activity of a polypeptide of the invention. Theinvention provides for the detection of aberrant expression of 4polypeptide of interest, comprising (a) assaying the expression of thepolypeptide of interest in cells or body fluid of an individual usingone or more antibodies specific to the polypeptide interest and (b)comparing the level of gene expression with a standard gene expressionlevel, whereby an increase or decrease in the assayed polypeptide geneexpression level compared to the standard expression level is indicativeof aberrant expression.

The invention provides a diagnostic assay for diagnosing diabetesmellitus, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of diabetesmellitus. With respect to insulin resistance, the presence of arelatively high or low amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of insulin resistance into diabetes mellitus.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol.101:976-985 (1985); Jalkanen et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One facet of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Each sequence isspecifically targeted to and can hybridize with a particular location onan individual human chromosome, thus each polynucleotide of the presentinvention can routinely be used as a chromosome marker using techniquesknown in the art. Table 1B.1, column 8 provides the chromosome locationof some of the polynucleotides of the invention.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably at least 15 bp (e.g., 15-25 bp) from the sequences shown inSEQ ID NO:X. Primers can optionally be selected using computer analysisso that primers do not span more than one predicted exon in the genomicDNA. These primers are then used for PCR screening of somatic cellhybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to SEQ ID NO:X will yield anamplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, preselection by hybridization to constructchromosome specific-cDNA libraries, and computer mapping techniques(See, e.g., Shuler, Trends Biotechnol 16:456459 (1998) which is herebyincorporated by reference in its entirety).

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).

Thus, the present invention also provides a method for chromosomallocalization which involves (a) preparing PCR primers from thepolynucleotide sequences in Table 1B and/or Table 2 and SEQ ID NO:X and(b) screening somatic cell hybrids containing individual chromosomes.

The polynucleotides of the present invention would likewise be usefulfor radiation hybrid mapping, HAPPY mapping, and long range restrictionmapping. For a review of these techniques and others known in the art,see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRL Press atOxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694(1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et al.,Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol.62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of whichis hereby incorporated by reference in its entirety.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library)). Column 9 of Table 1B.1 provides an OMIMreference identification number of diseases associated with thecytologic band disclosed in column 8 of Table 1B.1, as determined usingtechniques described herein and by reference to Table 5. Assuming 1megabase mapping resolution and one gene per 20 kb, a cDNA preciselylocalized to a chromosomal region associated with the disease could beone of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in a polynucleotideof the invention and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingthe polynucleotides of the invention. Any of these alterations (alteredexpression, chromosomal rearrangement, or mutation) can be used as adiagnostic or prognostic marker. Diagnostic and prognostic methods, kitsand reagents encompassed by the present invention are briefly describedbelow and more thoroughly elsewhere herein (see e.g., the sectionslabeled “Antibodies”, “Diagnostic Assays”, and “Methods for DetectingDiseases”).

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anindividual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder. Additional non-limiting examples of diagnosticmethods encompassed by the present invention are more thoroughlydescribed elsewhere herein (see, e.g., Example 12).

In still another embodiment, the invention includes a kit for analyzingsamples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the invention and a suitable container. In a specificembodiment, the kit includes two polynucleotide probes defining aninternal region of the polynucleotide of the invention, where each probehas one strand containing a 31′mer-end internal to the region. In afurther embodiment, the probes may be useful as primers for polymerasechain reaction amplification.

Where a diagnosis of a related disorder, including, for example,diagnosis of a tumor, has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced or depressed polynucleotide of theinvention expression will experience a worse clinical outcome relativeto patients expressing the gene at a level nearer the standard level.

By “measuring the expression level of polynucleotides of the invention”is intended qualitatively or quantitatively measuring or estimating thelevel of the polypeptide of the invention or the level of the mRNAencoding the polypeptide of the invention in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the related disorder orbeing determined by averaging levels from a population of individualsnot having a related disorder. As will be appreciated in the art, once astandard polypeptide level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains polypeptide of the present invention or the correspondingmRNA. As indicated, biological samples include body fluids (such assemen, lymph, vaginal pool, sera, plasma, urine, synovial fluid andspinal fluid) which contain the polypeptide of the present invention,and tissue sources found to express the polypeptide of the presentinvention. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides of theinvention are attached to a solid support. In one exemplary method, thesupport may be a “gene chip” or a “biological chip” as described in U.S.Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chipwith polynucleotides of the invention attached may be used to identifypolymorphisms between the isolated polynucleotide sequences of theinvention, with polynucleotides isolated from a test subject. Theknowledge of such polymorphisms (i.e. their location, as well as, theirexistence) would be beneficial in identifying disease loci for manydisorders, such as for example, in neural disorders, immune systemdisorders, muscular disorders, reproductive disorders, gastrointestinaldisorders, pulmonary disorders, digestive disorders, metabolicdisorders, cardiovascular disorders, renal disorders, proliferativedisorders, and/or cancerous diseases and conditions. Such a method isdescribed in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patentsreferenced supra are hereby incorporated by reference in their entiretyherein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides ofthe invention are incorporated onto a solid support, or gene chip. Forthe purposes of the present invention, a peptide nucleic acid (PNA) is apolyamide type of DNA analog and the monomeric units for adenine,guanine, thymine and cytosine are available commercially (PerceptiveBiosystems). Certain components of DNA, such as phosphorus, phosphorusoxides, or deoxyribose derivatives, are not present in PNAs. Asdisclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm etal., Nature 365, 666 (1993), PNAs bind specifically and tightly tocomplementary DNA strands and are not degraded by nucleases. In fact,PNA binds more strongly to DNA than DNA itself does. This is probablybecause there is no electrostatic repulsion between the two strands, andalso the polyamide backbone is more flexible. Because of this, PNA/DNAduplexes bind under a wider range of stringency conditions than DNA/DNAduplexes, making it easier to perform multiplex hybridization. Smallerprobes can be used than with DNA due to the strong binding. In addition,it is more likely that single base mismatches can be determined withPNA/DNA hybridization because a single mismatch in a PNA/DNA 15-merlowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for theDNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA meansthat hybridization can be done at low ionic strengths and reducepossible interference by salt during the analysis.

The compounds of the present invention have uses which include, but arenot limited to, detecting cancer in mammals. In particular the inventionis useful during diagnosis of pathological cell proliferative neoplasiaswhich include, but are not limited to: acute myelogenous leukemiasincluding acute monocytic leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute myelomonocytic leukemia, acuteerythroleukemia, acute megakaryocytic leukemia, and acuteundifferentiated leukemia, etc.; and chronic myelogenous leukemiasincluding chronic myelomonocytic leukemia, chronic granulocyticleukemia, etc. Preferred mammals include monkeys, apes, cats, dogs,cows, pigs, horses, rabbits and humans. Particularly preferred arehumans.

Pathological cell proliferative disorders are often associated withinappropriate activation of proto-oncogenes. (Gelmann, E. P. et al.,“The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,”in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds.,161-182 (1985)). Neoplasias are now believed to result from thequalitative alteration of a normal cellular gene product, or from thequantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocyticleukemia cell line HL-60. When HL-60 cells are chemically induced tostop proliferation, the level of c-myc is found to be downregulated.(International Publication Number WO 91/15580). However, it has beenshown that exposure of HL-60 cells to a DNA construct that iscomplementary to the 5′ end of c-myc or c-myb blocks translation of thecorresponding mRNAs which downregulates expression of the c-myc or c-mybproteins and causes arrest of cell proliferation and differentiation ofthe treated cells. (International Publication Number WO 91/15580;Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al.,Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisanwould appreciate the present invention's usefulness is not be limited totreatment, prevention, and/or prognosis of proliferative disorders ofcells and tissues of hematopoietic origin, in light of the numerouscells and cell types of varying origins which are known to exhibitproliferative phenotypes.

In addition to the foregoing, a polynucleotide of the present inventioncan be used to control gene expression through triple helix formation orthrough antisense DNA or RNA. Antisense techniques are discussed, forexample, in Okano, J. Neurochem. 56:560 (1991); “Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance Lee et al.,Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241: 456(1988); and Dervan et al., Science 251: 1360 (1991). Both methods relyon binding of the polynucleotide to a complementary DNA or RNA. Forthese techniques, preferred polynucleotides are usually oligonucleotides20 to 40 bases in length and complementary to either the region of thegene involved in transcription (triple helix—see Lee et al., Nucl. AcidsRes. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan etal., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. The oligonucleotide described above can also bedelivered to cells such that the antisense RNA or DNA may be expressedin vivo to inhibit production of polypeptide of the present inventionantigens. Both techniques are effective in model systems, and theinformation disclosed herein can be used to design antisense or triplehelix polynucleotides in an effort to treat disease, and in particular,for the treatment of proliferative diseases and/or conditions.Non-limiting antisense and triple helix methods encompassed by thepresent invention are more thoroughly described elsewhere herein (see,e.g., the section labeled “Antisense and Ribozyme (Antagonists)”).

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. Additionalnon-limiting examples of gene therapy methods encompassed by the presentinvention are more thoroughly described elsewhere herein (see, e.g., thesections labeled “Gene Therapy Methods”, and Examples 16, 17 and 18).

The polynucleotides are also useful for identifying individuals fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an individual's genome. These sequences can beused to prepare PCR primers for amplifying and isolating such selectedDNA, which can then be sequenced. Using this technique, individuals canbe identified because each individual will have a unique set of DNAsequences. Once an unique ID database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk,lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can beamplified using PCR. In one prior art technique, gene sequencesamplified from polymorphic loci, such as DQa class II HLA gene, are usedin forensic biology to identify individuals. (Erlich, H., PCRTechnology, Freeman and Co. (1992)). Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,polynucleotides of the present invention can be used as polymorphicmarkers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers prepared from the sequencesof the present invention, specific to tissues, including but not limitedto those shown in Table 1B. Panels of such reagents can identify tissueby species and/or by organ type. In a similar fashion, these reagentscan be used to screen tissue cultures for contamination. Additionalnon-limiting examples of such uses are further described herein.

The polynucleotides of the present invention are also useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to polypeptides of the present invention are usefulto provide immunological probes for differential identification of thetissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g.,immunocytochemistry assays). In addition, for a number of disorders ofthe above tissues or cells, significantly higher or lower levels of geneexpression of the polynucleotides/polypeptides of the present inventionmay be detected in certain tissues (e.g., tissues expressingpolypeptides and/or polynucleotides of the present invention, forexample, those disclosed in Table 1B, and/or cancerous and/or woundedtissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum,plasma, urine, synovial fluid or spinal fluid) taken from an individualhaving such a disorder, relative to a “standard” gene expression level,i.e., the expression level in healthy tissue from an individual nothaving the disorder.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves: (a) assaying gene expression level in cells or body fluid ofan individual; (b) comparing the gene expression level with a standardgene expression level, whereby an increase or decrease in the assayedgene expression level compared to the standard expression level isindicative of a disorder.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

Polypeptides and antibodies directed to polypeptides of the presentinvention are useful to provide immunological probes for differentialidentification of the tissue(s) (e.g., immunohistochemistry assays suchas, for example, ABC immunoperoxidase (Hsu et al., J. Histochem.Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistryassays).

Antibodies can be used to assay levels of polypeptides encoded bypolynucleotides of the invention in a biological sample using classicalimmunohistological methods known to those of skill in the art (e.g., seeJalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al.,J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methodsuseful for detecting protein gene expression include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase; radioisotopes,such as iodine (¹³¹I, ¹²⁵i, ¹²³i, ¹²¹j, carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), andtechnetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹pm, ¹⁴⁰La, ¹⁷⁵yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying levels of polypeptide of the present inventionin a biological sample, proteins can also be detected in vivo byimaging. Antibody labels or markers for in vivo imaging of proteininclude those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I, carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In,¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe),fluorine (¹⁸F, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously or intraperitoneally) into themammal to be examined for immune system disorder. It will be understoodin the art that the size of the subject and the imaging system used willdetermine the quantity of imaging moiety needed to produce diagnosticimages. In the case of a radioisotope moiety, for a human subject, thequantity of radioactivity injected will normally range from about 5 to20 millicuries of ^(99m)Tc. The labeled antibody or antibody fragmentwill then preferentially accumulate at the location of cells whichexpress the polypeptide encoded by a polynucleotide of the invention. Invivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., polypeptides encoded bypolynucleotides of the invention and/or antibodies) that are associatedwith heterologous polypeptides or nucleic acids. In one example, theinvention provides a method for delivering a therapeutic protein intothe targeted cell. In another example, the invention provides a methodfor delivering a single stranded nucleic acid (e.g., antisense orribozymes) or double stranded nucleic acid (e.g., DNA that can integrateinto the cell's genome or replicate episomally and that can betranscribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention in association with toxinsor cytotoxic prodrugs.

By “toxin” is meant one or more compounds that bind and activateendogenous cytotoxic effector systems, radioisotopes, holotoxins,modified toxins, catalytic subunits of toxins, or any molecules orenzymes not normally present in or on the surface of a cell that underdefined conditions cause the cell's death. Toxins that may be usedaccording to the methods of the invention include, but are not limitedto, radioisotopes known in the art, compounds such as, for example,antibodies (or complement fixing containing portions thereof) that bindan inherent or induced endogenous cytotoxic effector system, thymidinekinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonasexotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweedantiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includesa cytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or otherradioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³³I, ⁶⁸Ge, ⁵⁷Co,⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸rhenium; luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. In a specific embodiment, the invention providesa method for the specific destruction of cells (e.g., the destruction oftumor cells) by administering polypeptides of the invention orantibodies of the invention in association with the radioisotope ⁹⁰Y. Inanother specific embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹¹¹In. In a furtherspecific embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention or antibodies of theinvention in association with the radioisotope ¹³¹I.

Techniques known in the art may be applied to label polypeptides of theinvention (including antibodies). Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety).

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression level of a polypeptide of thepresent invention in cells or body fluid of an individual; and (b)comparing the assayed polypeptide expression level with a standardpolypeptide expression level, whereby an increase or decrease in theassayed polypeptide expression level compared to the standard expressionlevel is indicative of a disorder. With respect to cancer, the presenceof a relatively high amount of transcript in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat orprevent diseases or conditions such as, for example, neural disorders,immune system disorders, muscular disorders, reproductive disorders,gastrointestinal disorders, pulmonary disorders, cardiovasculardisorders, renal disorders, proliferative disorders, and/or cancerousdiseases and conditions. For example, patients can be administered apolypeptide of the present invention in an effort to replace absent ordecreased levels of the polypeptide (e.g., insulin), to supplementabsent or decreased levels of a different polypeptide (e.g., hemoglobinS for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit theactivity of a polypeptide (e.g., an oncogene or tumor supressor), toactivate the activity of a polypeptide (e.g., by binding to a receptor),to reduce the activity of a membrane bound receptor by competing with itfor free ligand (e.g., soluble TNF receptors used in reducinginflammation), or to bring about a desired response (e.g., blood vesselgrowth inhibition, enhancement of the immune response to proliferativecells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat disease (as described supra, and elsewhereherein). For example, administration of an antibody directed to apolypeptide of the present invention can bind, and/or neutralize thepolypeptide, and/or reduce overproduction of the polypeptide. Similarly,administration of an antibody can activate the polypeptide, such as bybinding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the biological activitiesdescribed herein.

Diagnostic Assays

The compounds of the present invention are useful for diagnosis,treatment, prevention and/or prognosis of various disorders in mammals,preferably humans. Such disorders include, but are not limited to, thoserelated to biological activities described in Table 1D and, also asdescribed herein under the section heading “Biological Activities”.

For a number of disorders, substantially altered (increased ordecreased) levels of gene expression can be detected in tissues, cellsor bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid orspinal fluid) taken from an individual having such a disorder, relativeto a “standard” gene expression level, that is, the expression level intissues or bodily fluids from an individual not having the disorder.Thus, the invention provides a diagnostic method useful during diagnosisof a disorder, which involves measuring the expression level of the geneencoding the polypeptide in tissues, cells or body fluid from anindividual and comparing the measured gene expression level with astandard gene expression level, whereby an increase or decrease in thegene expression level(s) compared to the standard is indicative of adisorder. These diagnostic assays may be performed in vivo or in vitro,such as, for example, on blood samples, biopsy tissue or autopsy tissue.

The present invention is also useful as a prognostic indicator, wherebypatients exhibiting enhanced or depressed gene expression willexperience a worse clinical outcome relative to patients expressing thegene at a level nearer the standard level.

In certain embodiments, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to diagnose and/or prognosticate diseasesand/or disorders associated with the tissue(s) in which the polypeptideof the invention is expressed, including one, two, three, four, five, ormore tissues disclosed in Table 1B.2. column 5 (Tissue DistributionLibrary Code).

By “assaying the expression level of the gene encoding the polypeptide”is intended qualitatively or quantitatively measuring or estimating thelevel of the polypeptide of the invention or the level of the mRNAencoding the polypeptide of the invention in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide expression level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardpolypeptide level or mRNA level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once astandard polypeptide level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source containingpolypeptides of the invention (including portions thereof) or mRNA. Asindicated, biological samples include body fluids (such as sera, plasma,urine, synovial fluid and spinal fluid) and tissue sources found toexpress the full length or fragments thereof of a polypeptide or mRNA.Methods for obtaining tissue biopsies and body fluids from mammals arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the polypeptides of the invention are then assayed usingany appropriate method. These include Northern blot analysis, S1nuclease mapping, the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction(RT-PCR), and reverse transcription in combination with the ligase chainreaction (RT-LCR).

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of polypeptidesof the invention, in a biological sample (e.g., cells and tissues),including determination of normal and abnormal levels of polypeptides.Thus, for instance, a diagnostic assay in accordance with the inventionfor detecting over-expression of polypeptides of the invention comparedto normal control tissue samples may be used to detect the presence oftumors. Assay techniques that can be used to determine levels of apolypeptide, such as a polypeptide of the present invention in a samplederived from a host are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays. Assaying polypeptide levels in abiological sample can occur using any art-known method.

Assaying polypeptide levels in a biological sample can occur usingantibody-based techniques. For example, polypeptide expression intissues can be studied with classical immunohistological methods(Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., etal., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methodsuseful for detecting polypeptide gene expression include inmmunoassays,such as the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I, carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the gene of inteest (such as,for example, cancer). The protein isolation methods employed herein may,for example, be such as those described in Harlow and Lane (Harlow, E.and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cellsthat could be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thegene.

For example, antibodies, or fragments of antibodies, such as thosedescribed herein, may be used to quantitatively or qualitatively detectthe presence of gene products or conserved variants or peptide fragmentsthereof. This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody coupled with lightmicroscopic, flow cytometric, or fluorimetric detection.

In a preferred embodiment, antibodies, or fragments of antibodiesdirected to any one or all of the predicted epitope domains of thepolypeptides of the invention (shown in Table 1B) may be used toquantitatively or qualitatively detect the presence of gene products orconserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody coupled with light microscopic, flowcytometric, or fluorimetric detection.

In an additional preferred embodiment, antibodies, or fragments ofantibodies directed to a conformational epitope of a polypeptide of theinvention may be used to quantitatively or qualitatively detect thepresence of gene products or conserved variants or peptide fragmentsthereof. This can be accomplished, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody coupled with lightmicroscopic, flow cytometric, or fluorimetric detection.

The antibodies (or fragments thereof), and/or polypeptides of thepresent invention may, additionally, be employed histologically, as inimmunofluorescence, immunoelectron microscopy or non-immunologicalassays, for in situ detection of gene products or conserved variants orpeptide fragments thereof. In situ detection may be accomplished byremoving a histological specimen from a patient, and applying thereto alabeled antibody or polypeptide of the present invention. The antibody(or fragment thereof) or polypeptide is preferably applied by overlayingthe labeled antibody (or fragment) onto a biological sample. Through theuse of such a procedure, it is possible to determine not only thepresence of the gene product, or conserved variants or peptidefragments, or polypeptide binding, but also its distribution in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Immunoassays and non-immunoassays for gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding gene products or conserved variants or peptide fragmentsthereof, and detecting the bound antibody by any of a number oftechniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled antibody or detectablepolypeptide of the invention. The solid phase support may then be washedwith the buffer a second time to remove unbound antibody or polypeptide.Optionally the antibody is subsequently labeled. The amount of boundlabel on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody or antigen polypeptidemay be determined according to well known methods. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

In addition to assaying polypeptide levels or polynucleotide levels in abiological sample obtained from an individual, polypeptide orpolynucleotide can also be detected in vivo by imaging. For example, inone embodiment of the invention, polypeptides and/or antibodies of theinvention are used to image diseased cells, such as neoplasms. Inanother embodiment, polynucleotides of the invention (e.g.,polynucleotides complementary to all or a portion of an mRNA) and/orantibodies (e.g., antibodies directed to any one or a combination of theepitopes of a polypeptide of the invention, antibodies directed to aconformational epitope of a polypeptide of the invention, or antibodiesdirected to the full length polypeptide expressed on the cell surface ofa mammalian cell) are used to image diseased or neoplastic cells.

Antibody labels or markers for in vivo imaging of polypeptides of theinvention include those detectable by X-radiography, NMR, MRI, CAT-scansor ESR. For X-radiography, suitable labels include radioisotopes such asbarium or cesium, which emit detectable radiation but are not overtlyharmful to the subject. Suitable markers for NMR and ESR include thosewith a detectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma. Where in vivo imaging is used to detect enhanced levels ofpolypeptides for diagnosis in humans, it may be preferable to use humanantibodies or “humanized” chimeric monoclonal antibodies. Suchantibodies can be produced using techniques described herein orotherwise known in the art. For example methods for producing chimericantibodies are known in the art. See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al, Nature 314:268(1985).

Additionally, any polypeptides of the invention whose presence can bedetected, can be administered. For example, polypeptides of theinvention labeled with a radio-opaque or other appropriate compound canbe administered and visualized in vivo, as discussed, above for labeledantibodies. Further, such polypeptides can be utilized for in vitrodiagnostic procedures.

A polypeptide-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for a disorder. Itwill be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodyor antibody fragment will then preferentially accumulate at the locationof cells which contain the antigenic protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

With respect to antibodies, one of the ways in which an antibody of thepresent invention can be detectably labeled is by linking the same to areporter enzyme and using the linked product in an enzyme immunoassay(EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”,1978, Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.); Voller et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981);Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton,Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, KgakuShoin, Tokyo). The reporter enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Reporter enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for thereporter enzyme. Detection may also be accomplished by visual comparisonof the extent of enzymatic reaction of a substrate in comparison withsimilarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect polypeptides through theuse of a radioimmunoassay (RIA) (see, for example, Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986, which isincorporated by reference herein). The radioactive isotope can bedetected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Methods for Detecting Diseases

In general, a disease may be detected in a patient based on the presenceof one or more proteins of the invention and/or polynucleotides encodingsuch proteins in a biological sample (for example, blood, sera, urine,and/or tumor biopsies) obtained from the patient. In other words, suchproteins may be used as markers to indicate the presence or absence of adisease or disorder, including cancer and/or as described elsewhereherein. In addition, such proteins may be useful for the detection ofother diseases and cancers. The binding agents provided herein generallypermit detection of the level of antigen that binds to the agent in thebiological sample. Polynucleotide primers and probes may be used todetect the level of mRNA encoding polypeptides of the invention, whichis also indicative of the presence or absence of a disease or disorder,including cancer. In general, polypeptides of the invention should bepresent at a level that is at least three fold higher in diseased tissuethan in normal tissue.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, supra. In general, the presence orabsence of a disease in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of a bindingagent(s) immobilized on a solid support to bind to and remove thepolypeptide of the invention from the remainder of the sample. The boundpolypeptide may then be detected using a detection reagent that containsa reporter group and specifically binds to the binding agent/polypeptidecomplex. Such detection reagents may comprise, for example, a bindingagent that specifically binds to the polypeptide or an antibody or otheragent that specifically binds to the binding agent, such as ananti-immunoglobulin, protein G, protein A or a lectin. Alternatively, acompetitive assay may be utilized, in which a polypeptide is labeledwith a reporter group and allowed to bind to the immobilized bindingagent after incubation of the binding agent with the sample. The extentto which components of the sample inhibit the binding of the labeledpolypeptide to the binding agent is indicative of the reactivity of thesample with the immobilized binding agent. Suitable polypeptides for usewithin such assays include polypeptides of the invention and portionsthereof, or antibodies, to which the binding agent binds, as describedabove.

The solid support may be any material known to those of skill in the artto which polypeptides of the invention may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for the suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 ug, andpreferably about 100 ng to about 1 ug, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

Gene Therapy Methods

Also encompassed by the invention are gene therapy methods for treatingor preventing disorders, diseases and conditions. The gene therapymethods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofthe polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the present inventionoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the present invention ex vivo, with the engineeredcells then being provided to a patient to be treated with thepolypeptide of the present invention. Such methods are well-known in theart. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst.85:207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112(1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994);Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al.,Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., HumanGene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3:31-38 (1996)), which are herein incorporated by reference. In oneembodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the present invention isdelivered as a naked polynucleotide. The term “naked” polynucleotide,DNA or RNA refers to sequences that are free from any delivery vehiclethat acts to assist, promote or facilitate entry into the cell,including viral sequences, viral particles, liposome formulations,lipofectin or precipitating agents and the like. However, thepolynucleotide of the present invention can also be delivered inliposome formulations and lipofectin formulations and the like can beprepared by methods well known to those skilled in the art. Such methodsare described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Appropriatevectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; andpEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Othersuitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of the polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for the polynucleotide of the presentinvention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs are complexed in aliposome preparation. Liposomal preparations for use in the instantinvention include cationic (positively charged), anionic (negativelycharged) and neutral preparations. However, cationic liposomes areparticularly preferred because a tight charge complex can be formedbetween the cationic liposome and the polyanionic nucleic acid. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416,which is herein incorporated by reference); mRNA (Malone et al., Proc.Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporatedby reference); and purified transcription factors (Debs et al., J. Biol.Chem. (1990) 265:10189-10192, which is herein incorporated byreference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15 EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys.Res. Commun. 76:836 (1977); Fraley et al., Proc. Nat. Acad. Sci. USA76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978);Schaefer-Ridder et al., Science 215:166 (1982)), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 provide methodsfor delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodinga polypeptide of the present invention. Retroviruses from which theretroviral plasmid vectors may be derived include, but are not limitedto, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus,and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E−86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding a polypeptide of the presentinvention. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express a polypeptide of the present invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotide contained in an adenovirus vector. Adenovirus can bemanipulated such that it encodes and expresses a polypeptide of thepresent invention, and at the same time is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. Adenovirusexpression is achieved without integration of the viral DNA into thehost cell chromosome, thereby alleviating concerns about insertionalmutagenesis. Furthermore, adenoviruses have been used as live entericvaccines for many years with an excellent safety profile (Schwartz etal. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirusmediated gene transfer has been demonstrated in a number of instancesincluding transfer of alpha-1-antitrypsin and CFTR to the lungs ofcotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434;Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691692 (1993);and U.S. Pat. No. 5,652,224, which are herein incorporated by reference.For example, the adenovirus vector Ad2 is useful and can be grown inhuman 293 cells. These cells contain the E1region of adenovirus andconstitutively express E1a and E1b, which complement the defectiveadenoviruses by providing the products of the genes deleted from thevector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3,Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructis inserted into the AAV vector using standard cloning methods, such asthose found in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (1989). The recombinant AAV vector is thentransfected into packaging cells which are infected with a helper virus,using any standard technique, including lipofection, electroporation,calcium phosphate precipitation, etc. Appropriate helper viruses includeadenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.Once the packaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct. These viral particles are then used to transduce eukaryoticcells, either ex vivo or in vivo. The transduced cells will contain thepolynucleotide construct integrated into its genome, and will express apolypeptide of the invention.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding a polypeptide of the present invention) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), which are hereinencorporated by reference. This method involves the activation of a genewhich is present in the target cells, but which is not normallyexpressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotide encoding a polypeptide of the present invention maycontain a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a proteinoated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site. In specificembodiments, suitable delivery vehicles for use with systemicadministration comprise liposomes comprising polypeptides of theinvention for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, can be used in assays to test for one or morebiological activities. If these polynucleotides or polypeptides, oragonists or antagonists of the present invention, do exhibit activity ina particular assay, it is likely that these molecules may be involved inthe diseases associated with the biological activity. Thus, thepolynucleotides and polypeptides, and agonists or antagonists could beused to treat the associated disease.

Members of the secreted family of proteins are believed to be involvedin biological activities associated with, for example, cellularsignaling. Accordingly, compositions of the invention (includingpolynucleotides, polypeptides and antibodies of the invention, andfragments and variants thereof) may be used in diagnosis, prognosis,prevention and/or treatment of diseases and/or disorders associated withaberrant activity of secreted polypeptides.

In preferred embodiments, compositions of the invention (includingpolynucleotides, polypeptides and antibodies of the invention, andfragments and variants thereof) may be used in the diagnosis, prognosis,prevention, treatment, and/or amelioration of diseases and/or disordersrelating to diabetes mellitus and/or a condition associated withdiabetes mellitus (e.g., hyperglycemia, obesity, diabetic refinopathy,mononeuropathy, polyneuropathy, atherosclerosis, ulcers, heart disease,anemia, stroke, gangrene (e.g., of the feet and hands), impotence,infection, cataract, poor kidney function, malfunctioning of theautonomic nervous system, impaired white blood cell function, Carpaltunnel syndrome, Dupuytren's contracture, diabetic ketoacidosis, and asdescribed in “Renal Disorders”, “Wound Healing and Epithelial CellProliferation”, “Endocrine Disorders”, “Reproductive System Disorders”,and “Gastrointestinal Disorders” sections below).

In certain embodiments, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to diagnose and/or prognosticate diseasesand/or disorders associated with the tissue(s) in which the polypeptideof the invention is expressed including one, two, three, four, five, ormore tissues disclosed in Table 1B.2, column 5 (Tissue DistributionLibrary Code).

Thus, polynucleotides, translation products and antibodies of theinvention are useful in the diagnosis, detection, prevention,prognistication, and/or treatment of diseases and/or disordersassociated with activities that include, but are not limited to,prohormone activation, neurotransmitter activity, cellular signaling,cellular proliferation, cellular differentiation, and cell migration.

More generally, polynucleotides, translation products and antibodiescorresponding to this gene may be useful for the diagnosis, prognosis,prevention, treatment and/or amelioration of diseases and/or disordersassociated with the following system or systems.

Renal Disorders

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention, may be used to detect, prevent,diagnose, prognosticate, treat, and/or ameliorate disorders of the renalsystem. Renal disorders which can be detected, prevented, diagnosed,prognosticated, treated, and/or ameliorated with compositions of theinvention include, but are not limited to, kidney failure, nephritis,blood vessel disorders of kidney, metabolic and congenital kidneydisorders, urinary disorders of the kidney, autoimmune disorders,sclerosis and necrosis, electrolyte imbalance, and kidney cancers.

Kidney diseases which can be detected, prevented, diagnosed,prognosticated, treated, and/or ameliorated with compositions of theinvention include, but are not limited to, acute kidney failure, chronickidney failure, atheroembolic renal failure, end-stage renal disease,inflammatory diseases of the kidney (e.g., acute glomerulonephritis,postinfectious glomerulonephritis, rapidly progressiveglomerulonephritis, nephrotic syndrome, membranous glomerulonephritis,familial nephrotic syndrome, membranoproliferative glomerulonephritis Iand II, mesangial proliferative glomerulonephritis, chronicglomerulonephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, acute post-streptococcalglomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronicnephritis, interstitial nephritis, and post-streptococcalglomerulonephritis), blood vessel disorders of the kidneys (e.g., kidneyinfarction, atheroembolic kidney disease, cortical necrosis, malignantnephrosclerosis, renal vein thrombosis, renal underperfusion, renalretinopathy, renal ischemia-reperfusion, renal artery embolism, andrenal artery stenosis), and kidney disorders resulting form urinarytract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renallithiasis, nephrolithiasis), reflux nephropathy, urinary tractinfections, urinary retention, and acute or chronic unilateralobstructive uropathy.)

In addition, compositions of the invention can be used to detect,prevent, diagnose, prognosticate, treat, and/or ameliorate metabolic andcongenital disorders of the kidney (e.g., uremia, renal amyloidosis,renal osteodystrophy, renal tubular acidosis, renal glycosuria,nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renalfibrocystic osteosis (renal rickets), Hartnup disease, Bartter'ssyndrome, Liddle's syndrome, polycystic kidney disease, medullary cysticdisease, medullary sponge kidney, Alport's syndrome, nail-patellasyndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoekidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesicnephropathy, kidney stones, and membranous nephropathy), and autoimmunedisorders of the kidney (e.g., systemic lupus erythematosus (SLE),Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferativeglomerulonephritis).

Compositions of the invention can also be used to detect, prevent,diagnose, prognosticate, treat, and/or ameliorate sclerotic or necroticdisorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy,focal segmental glomerulosclerosis (FSGS), necrotizingglomerulonephritis, and renal papillary necrosis), cancers of the kidney(e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer,transitional cell cancer, renal adenocarcinoma, squamous cell cancer,and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis,pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypematremia,hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia,hypophosphatemia, and hyperphosphatemia).

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides may be administered as part of a Therapeutic,described in more detail below. Methods of delivering polynucleotidesare described in more detail herein.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associated with systemic treatment with steroids,radiation therapy and antineoplastic drugs and antimetabolites.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could be used to promote dermal reestablishmentsubsequent to dermal loss

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that polynucleotides orpolypeptides, agonists or antagonists of the present invention, could beused to increase adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermiic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could promote proliferation of epithelial cellssuch as sebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. Polynucleotides or polypeptides, agonists or antagonists of thepresent invention, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, may have a cytoprotectiveeffect on the small intestine mucosa. Polynucleotides or polypeptides,as well as agonists or antagonists of the present invention, may alsostimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could further be used in full regeneration ofskin in full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. Polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, could also be used to treatgastric and doudenal ulcers and help heal by scar formation of themucosal lining and regeneration of glandular mucosa and duodenal mucosallining more rapidly. Inflammatory bowel diseases, such as Crohn'sdisease and ulcerative colitis, are diseases which result in destructionof the mucosal surface of the small or large intestine, respectively.Thus, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent progression of inflammatory bowel disease. Treatment withpolynucleotides or polypeptides, agonists or antagonists of the presentinvention, is expected to have a significant effect on the production ofmucus throughout the gastrointestinal tract and could be used to protectthe intestinal mucosa from injurious substances that are ingested orfollowing surgery. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, could be used to treat diseasesassociate with the under expression.

Moreover, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to prevent and healdamage to the lungs due to various pathological states. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing polynucleotides or polypeptides, agonists or antagonists of thepresent invention. Also, polynucleotides or polypeptides, as well asagonists or antagonists of the present invention, could be used tostimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

In addition, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andH diabetes, where some islet cell function remains, polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, could be used to maintain the islet function so as toalleviate, delay or prevent permanent manifestation of the disease.Also, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used as an auxiliary inislet cell transplantation to improve or promote islet cell function.

Endocrine Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, may be used to detect, prevent, diagnose,prognosticate, treat, and/or ameliorate disorders and/or diseasesrelated to hormone imbalance, and/or disorders or diseases of theendocrine system.

Hormones secreted by the glands of the endocrine system control physicalgrowth, sexual function, metabolism, and other functions. Disorders maybe classified in two ways: disturbances in the production of hormones,and the inability of tissues to respond to hormones. The etiology ofthese hormone imbalance or endocrine system diseases, disorders orconditions may be genetic, somatic, such as cancer and some autoimmunediseases, acquired (e.g., by chemotherapy, injury or toxins), orinfectious. Moreover, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention can be used as a markeror detector of a particular disease or disorder related to the endocrinesystem and/or hormone imbalance.

Endocrine system and/or hormone imbalance and/or diseases encompassdisorders of uterine motility including, but not limited to:complications with pregnancy and labor (e.g., pre-term labor, post-termpregnancy, spontaneous abortion, and slow or stopped labor); anddisorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea andendometriosis).

Endocrine system and/or hormone imbalance disorders and/or diseasesinclude disorders and/or diseases of the pancreas, such as, for example,diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis,pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases ofthe adrenal glands such as, for example, Addison's Disease,corticosteroid deficiency, virilizing disease, hirsutism, Cushing'sSyndrome, hyperaldosteronism, pheochromocytoma; disorders and/ordiseases of the pituitary gland, such as, for example, hyperpituitarism,hypopituitarism, pituitary dwarfism, pituitary adenoma,panhypopituitarism, acromegaly, gigantism; disorders and/or diseases ofthe thyroid, including but not limited to, hyperthyroidism,hypothyroidism, Plummer's disease, Graves' disease (toxic diffusegoiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis,subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis),Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormonecoupling defect, thymic aplasia, Hurthle cell tumours of the thyroid,thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma;disorders and/or diseases of the parathyroid, such as, for example,hyperparathyroidism, hypoparathyroidism; disorders and/or diseases ofthe hypothalamus.

In addition, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases of the testes orovaries, including cancer. Other disorders and/or diseases of the testesor ovaries further include, for example, ovarian cancer, polycysticovary syndrome, Klinefelter's syndrome, vanishing testes syndrome(bilateral anorchia), congenital absence of Leydig's cells,cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillaryhaemangioma of the testis (benign), neoplasias of the testis andneo-testis.

Moreover, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases such as, forexample, polyglandular deficiency syndromes, pheochromocytoma,neuroblastoma, multiple Endocrine neoplasia, and disorders and/orcancers of endocrine tissues.

In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to detect, prevent, diagnose,prognosticate, treat, and/or ameliorate endocrine diseases and/ordisorders associated with the tissue(s) in which the polypeptide of theinvention is expressed, including one, two, three, four, five, or moretissues disclosed in Table 1B.2, column 5 (Tissue Distribution LibraryCode).

Reproductive System Disorders

The polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used for the detection, prevention, diagnosis,prognostication, treatment, and/or amelioration of diseases and/ordisorders of the reproductive system. Reproductive system disorders thatcan be treated by the compositions of the invention, include, but arenot limited to, reproductive system injuries, infections, neoplasticdisorders, congenital defects, and diseases or disorders which result ininfertility, complications with pregnancy, labor, or parturition, andpostpartum difficulties.

Reproductive system disorders and/or diseases include diseases and/ordisorders of the testes, including testicular atrophy, testicularfeminization, cryptorchism (unilateral and bilateral), anorchia, ectopictestis, epididymitis and orchitis (typically resulting from infectionssuch as, for example, gonorrhea, mumps, tuberculosis, and syphilis),testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas,embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sactumors, and teratomas), stromal tumors (e.g., Leydig cell tumors),hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, anddisorders of sperm production (e.g., immotile cilia syndrome, aspermia,asthenozoospermia, azoosperrnia, oligospermia, and teratozoospermia).

Reproductive system disorders also include disorders of the prostategland, such as acute non-bacterial prostatitis, chronic non-bacterialprostatitis, acute bacterial prostatitis, chronic bacterial prostatitis,prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia,benign prostatic hypertrophy or hyperplasia, and prostate neoplasticdisorders, including adenocarcinomas, transitional cell carcinomas,ductal carcinomas, and squamous cell carcinomas.

Additionally, the compositions of the invention may be useful in thedetection, prevention, diagnosis, prognostication, treatment, and/oramelioration of disorders or diseases of the penis and urethra,including inflammatory disorders, such as balanoposthitis, balanitisxerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplexvirus, gonorrhea, non-gonococcal urethritis, chiamydia, mycoplasma,trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum,condyloma latum, and pearly penile papules; urethral abnormalities, suchas hypospadias, epispadias, and phimosis; premalignant lesions,including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis,giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penilecancers, including squamous cell carcinomas, carcinoma in situ,verrucous carcinoma, and disseminated penile carcinoma; urethralneoplastic disorders, including penile urethral carcinoma,bulbomembranous urethral carcinoma, and prostatic urethral carcinoma;and erectile disorders, such as priapism, Peyronie's disease, erectiledysfunction, and impotence.

Moreover, diseases and/or disorders of the vas deferens includevasculititis and CBAVD (congenital bilateral absence of the vasdeferens); additionally, the polynucleotides, polypeptides, and agonistsor antagonists of the present invention may be used in the detection,prevention, diagnosis, prognostication, treatment, and/or ameliorationof diseases and disorders of the seminal vesicles, including hydatiddisease, congenital chloride diarrhea, and polycystic kidney disease.

Other disorders and/or diseases of the male reproductive system include,for example, Klinefelter's syndrome, Young's syndrome, prematureejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome,high fever, multiple sclerosis, and gynecomastia.

Further, the polynucleotides, polypeptides, and agonists or antagonistsof the present invention may be used in the detection, prevention,diagnosis, prognostication, treatment, and/or amelioration of diseasesand/or disorders of the vagina and vulva, including bacterial vaginosis,candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale,lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma,vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonasvaginitis, condyloma acuminatum, syphilis, molluscum contagiosum,atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus,vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvarvestibulitis, and neoplastic disorders, such as squamous cellhyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas,cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.

Disorders and/or diseases of the uterus include dysmenorrhea,retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatorybleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman'ssyndrome, premature menopause, precocious puberty, uterine polyps,dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals),and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, andsarcomas. Additionally, the polypeptides, polynucleotides, or agonistsor antagonists of the invention may be useful as a marker or detectorof, as well as in the detection, prevention, diagnosis, prognostication,treatment, and/or amelioration of congenital uterine abnormalities, suchas bicomuate uterus, septate uterus, simple unicornuate uterus,unicomuate uterus with a noncavitary rudimentary horn, unicornuateuterus with a non-communicating cavitary rudimentary horn, unicomuateuterus with a communicating cavitary horn, arcuate uterus, uterinedidelfus, and T-shaped uterus.

Ovarian diseases and/or disorders include anovulation, polycystic ovarysyndrome (Stein-Leventhal syndrome), ovarian cysts, ovarianhypofunction, ovarian insensitivity to gonadotropins, ovarianoverproduction of androgens, right ovarian vein syndrome, amenorrhea,hirutism, and ovarian cancer (including, but not limited to, primary andsecondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinomaof the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinousadenocarcinoma, and Ovarian Krukenberg tumors).

Cervical diseases and/or disorders include cervicitis, chroniccervicitis, mucopurulent cervicitis, cervical dysplasia, cervicalpolyps, Nabothian cysts, cervical erosion, cervical incompetence, andcervical neoplasms (including, for example, cervical carcinoma, squamousmetaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, andcolumnar cell neoplasia).

Additionally, diseases and/or disorders of the reproductive systeminclude disorders and/or diseases of pregnancy, including miscarriageand stillbirth, such as early abortion, late abortion, spontaneousabortion, induced abortion, therapeutic abortion, threatened abortion,missed abortion, incomplete abortion, complete abortion, habitualabortion, missed abortion, and septic abortion; ectopic pregnancy,anemia, Rh incompatibility, vaginal bleeding during pregnancy,gestational diabetes, intrauterine growth retardation, polyhydramnios,HELLP syndrome, abruptio placentae, placenta previa, hyperemesis,preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy.Additionally, the polynucleotides, polypeptides, and agonists orantagonists of the present invention may be used in the detection,prevention, diagnosis, prognostication, treatment, and/or ameliorationof diseases that can complicate pregnancy, including heart disease,heart failure, rheumatic heart disease, congenital heart disease, mitralvalve prolapse, high blood pressure, anemia, kidney disease, infectiousdisease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectioushepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus,Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis,chronic active hepatitis, cirrhosis of the liver, primary biliarycirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis,myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis,ovarian cysts, gallbladder disorders, and obstruction of the intestine.

Complications associated with labor and parturition include prematurerupture of the membranes, pre-term labor, post-term pregnancy,postmaturity, labor that progresses too slowly, fetal distress (e.g.,abnormal heart rate (fetal or maternal), breathing problems, andabnormal fetal position), shoulder dystocia, prolapsed umbilical cord,amniotic fluid embolism, and aberrant uterine bleeding.

Further, diseases and/or disorders of the postdelivery period, includingendometritis, myometritis, parametritis, peritonitis, pelvicthrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis,saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage,and inverted uterus.

Other disorders and/or diseases of the female reproductive system thatmay be detected, prevented, diagnosed, prognosticated, treated, and/orameliorated by the polynucleotides, polypeptides, and agonists orantagonists of the present invention include, for example, Turner'ssyndrome, pseudohermaphroditism, premenstrual syndrome, pelvicinflammatory disease, pelvic congestion (vascular engorgement),frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, andMittelschmerz.

Gastrointestinal Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, may be used to detect, prevent, diagnose,prognosticate, treat, and/or ameliorate gastrointestinal diseases anddisorders, including inflammatory diseases and/or conditions,infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of thesmall intestine, non-Hodgkin's lymphoma of the small intestine, smallbowl lymphoma)), and ulcers, such as peptic ulcers.

Gastrointestinal disorders include dysphagia, odynophagia, inflammationof the esophagus, peptic esophagitis, gastric reflux, submucosalfibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas,epidermal cancers, adeoncarcinomas, gastric retention disorders,gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of thestomach, autoimmune disorders such as pernicious anemia, pyloricstenosis, gastritis (bacterial, viral, eosinophilic, stress-induced,chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritonealdiseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis,neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).

Gastrointestinal disorders also include disorders associated with thesmall intestine, such as malabsorption syndromes, distension, irritablebowel syndrome, sugar intolerance, celiac disease, duodenal ulcers,duodenitis, tropical sprue, Whipple's disease, intestinallymphangiectasia, Crohn's disease, appendicitis, obstructions of theileum, Meckel's diverticulum, multiple diverticula, failure of completerotation of the small and large intestine, lymphoma, and bacterial andparasitic diseases (such as Traveler's diarrhea, typhoid andparatyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides),Hookworms (Ancylostoma duodenale), Threadworns (Enterobiusvermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus,Diphyllobothrium spp., and T. solium).

Liver diseases and/or disorders include intrahepatic cholestasis(alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholicfatty liver, reye syndrome), hepatic vein thrombosis, hepatolentriculardegeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenalsyndrome, portal hypertension (esophageal and gastric varices), liverabscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary andexperimental), alcoholic liver diseases (fatty liver, hepatitis,cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebicliver abscess), jaundice (hemolytic, hepatocellular, and cholestatic),cholestasis, portal hypertension, liver enlargement, ascites, hepatitis(alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune,hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis,viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitisD, hepatitis E), Wilson's disease, granulomatous hepatitis, secondarybiliary cirrhosis, hepatic encephalopathy, portal hypertension, varices,hepatic encephalopathy, primary biliary cirrhosis, primary sclerosingcholangitis, hepatocellular adenoma, hemangiomas, bile stones, liverfailure (hepatic encephalopathy, acute liver failure), and liverneoplasms (angiomyolipoma, calcified liver metastases, cystic livermetastases, epithelial tumors, fibrolamellar hepatocarcinoma, focalnodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma,hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liverhemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors ofliver, nodular regenerative hyperplasia, benign liver tumors (Hepaticcysts [Simple cysts, Polycystic liver disease, Hepatobiliarycystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymalhamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors[Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma),Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerativehyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi'ssarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosishepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittentporphyria, porphyria cutanea tarda), Zellweger syndrome).

Pancreatic diseases and/or disorders include acute pancreatitis, chronicpancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis),neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma,insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-celltumors, pancreoblastoma), and other pancreatic diseases (e.g., cysticfibrosis, cyst (pancreatic pseudocyst, pancreatic fistula,insufficiency)).

Gallbladder diseases include gallstones (cholelithiasis andcholedocholithiasis), postcholecystectomy syndrome, diverticulosis ofthe gallbladder, acute cholecystitis, chronic cholecystitis, bile ducttumors, and mucocele.

Diseases and/or disorders of the large intestine includeantibiotic-associated colitis, diverticulitis, ulcerative colitis,acquired megacolon, abscesses, fungal and bacterial infections,anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps(e.g., villous adenoma), colon carcinoma, colorectal cancer], colonicdiverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease,toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery),duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenalulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ilealdiseases (ileal neoplasms, ileitis), immunoproliferative smallintestinal disease, inflammatory bowel disease (ulcerative colitis,Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis,balantidiasis, blastocystis infections, cryptosporidiosis,dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula(rectal fistula), intestinal neoplasms (cecal neoplasms, colonicneoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps,jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferentloop syndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction [cecal volvulus], intussusception), intestinalperforation, intestinal polyps (colonic polyps, gardner syndrome,peutz-jeghers syndrome), jejunal diseases (ejunal neoplasms),malabsorption syndromes (blind loop syndrome, celiac disease, lactoseintolerance, short bowl syndrome, tropical sprue, whipple's disease),mesenteric vascular occlusion, pneumatosis cystoides intestinalis,protein-losing enteropathies (intestinal lymphagiectasis), rectaldiseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenalulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer,Zollinger-Ellison syndrome), postgastrectomy syndromes (dumpingsyndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux(bile reflux), gastric antral vascular ectasia, gastric fistula, gastricoutlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis,stomach dilatation, stomach diverticulum, stomach neoplasms (gastriccancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastricpolyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,postoperative nausea and vomiting) and hemorrhagic colitis.

Further diseases and/or disorders of the gastrointestinal system includebiliary tract diseases, such as, gastroschisis, fistula (e.g., biliaryfistula, esophageal fistula, gastric fistula, intestinal fistula,pancreatic fistula), neoplasms (e.g., biliary tract neoplasms,esophageal neoplasms, such as adenocarcinoma of the esophagus,esophageal squamous cell carcinoma, gastrointestinal neoplasms,pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinouscystic neoplasm of the pancreas, pancreatic cystic neoplasms,pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g.,bullous diseases, candidiasis, glycogenic acanthosis, ulceration,barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker'sdiverticulum), fistula (e.g., tracheoesophageal fistula), motilitydisorders (e.g., CREST syndrome, deglutition disorders, achalasia,spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaavesyndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatichernia (e.g., hiatal hernia); gastrointestinal diseases, such as,gastroenteritis (e.g., cholera morbus, norwalk virus infection),hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomachneoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma,stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoralhernia, inguinal hernia, obturator hernia, umbilical hernia, ventralhernia), and intestinal diseases (e.g., cecal diseases (appendicitis,cecal neoplasms)).

Chemotaxis

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treatwounds.

It is also contemplated that polynucleotides or polypeptides, as well asagonists or antagonists of the present invention may inhibit chemotacticactivity. These molecules could also be used to treat disorders. Thus,polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide. Preferred cells includecells from mammals, yeast, Drosophila, or E. coli. Cells expressing thepolypeptide (or cell membrane containing the expressed polypeptide) arethen preferably contacted with a test compound potentially containingthe molecule to observe binding, stimulation, or inhibition of activityof either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which the polypeptide of the presentinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of thepolypeptide of the present invention thereby effectively generatingagonists and antagonists of the polypeptide of the present invention.See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82(1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); andLorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each ofthese patents and publications are hereby incorporated by reference). Inone embodiment, alteration of polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired molecule byhomologous, or site-specific, recombination. In another embodiment,polynucleotides and corresponding polypeptides may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of the polypeptide of the present invention may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules are family members. Infurther preferred embodiments, the heterologous molecule is a growthfactor such as, for example, platelet-derived growth factor (PDGF),insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP4, BMP-5, BMP6,BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin,growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derivedneurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptide of the present invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide of the present invention. The biologicalactivity of the fragments may include an improved desired activity, or adecreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H{]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to a polypeptide of the invention comprising the steps of:(a) incubating a candidate binding compound with a polypeptide of thepresent invention; and (b) determining if binding has occurred.Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with a polypeptide of the present invention, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenylacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:X, or the complementary strand thereof, and/or to cDNA sequencescontained in cDNA ATCC Deposit No:Z identified for example, in Table 1Aand/or 1B. In one embodiment, antisense sequence is generatedinternally, by the organism, in another embodiment, the antisensesequence is separately administered (see, for example, O'Connor, J.,Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisensetechnology can be used to control gene expression through antisense DNAor RNA, or through triple-helix formation. Antisense techniques arediscussed for example, in Okano, J., Neurochem. 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooneyet al., Science 241:456 (1988); and Dervan et al., Science 251:1300(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the nonymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes thepolypeptide of the present invention may be used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription thereby preventing transcription and theproduction of the receptor. The antisense RNA oligonucleotide hybridizesto the mRNA in vivo and blocks translation of the mRNA molecule intoreceptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid. Such a vectorcan remain episomal or become chromosomally integrated, as long as itcan be transcribed to produce the desired antisense RNA. Such vectorscan be constructed by recombinant DNA technology methods standard in theart. Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in vertebrate cells. Expression of thesequence encoding the polypeptide of the present invention or fragmentsthereof, can be by any promoter known in the art to act in vertebrate,preferably human cells. Such promoters can be inducible or constitutive.Such promoters include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature 29:304-310 (1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner etal., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofthe present invention. However, absolute complementarity, althoughpreferred, is not required. A sequence “complementary to at least aportion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA it may contain and still form a stable duplex (ortriplex as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′ - or3′-non-translated, non-coding regions of polynucleotide sequencesdescribed herein could be used in an antisense approach to inhibittranslation of endogenous mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′-or coding region of mRNA of the present invention, antisensenucleic acids should be at least six nucleotides in length, and arepreferably oligonucleotides ranging from 6 to about 50 nucleotides inlength. In specific aspects the oligonucleotide is at least 10nucleotides, at least 17 nucleotides, at least 25 nucleotides or atleast 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the coding region sequencecould be used, those complementary to the transcribed untranslatedregion are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs, the use of hammerhead ribozymesis preferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within thenucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g., for improved stability,targeting, etc.) and should be delivered to cells which express in vivo.DNA constructs encoding the ribozyme may be introduced into the cell inthe same manner as described above for the introduction of antisenseencoding DNA. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous messages and inhibit translation. Sinceribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Thus, the invention provides a method of treating disorders or diseases,including but not limited to the disorders or diseases listed throughoutthis application, associated with overexpression of a polynucleotide ofthe present invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind polypeptides of theinvention, and the binding molecules identified thereby. These bindingmolecules are useful, for example, as agonists and antagonists of thepolypeptides of the invention. Such agonists and antagonists can beused, in accordance with the invention, in the therapeutic embodimentsdescribed in detail, below.

This method comprises the steps of:

-   -   contacting polypeptides of the invention with a plurality of        molecules; and    -   identifying a molecule that binds the polypeptides of the        invention.

The step of contacting the polypeptides of the invention with theplurality of molecules may be effected in a number of ways. For example,one may contemplate immobilizing the polypeptides on a solid support andbringing a solution of the plurality of molecules in contact with theimmobilized polypeptides. Such a procedure would be akin to an affinitychromatographic process, with the affinity matrix being comprised of theimmobilized polypeptides of the invention. The molecules having aselective affinity for the polypeptides can then be purified by affinityselection. The nature of the solid support, process for attachment ofthe polypeptides to the solid support, solvent, and conditions of theaffinity isolation or selection are largely conventional and well knownto those of ordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the polypeptides of the invention, optionally in thepresence of an inducer should one be required for expression, todetermine if any selective affinity interaction takes place between thepolypeptides and the individual clone. Prior to contacting thepolypeptides with each fraction comprising individual polypeptides, thepolypeptides could first be transferred to a solid support foradditional convenience. Such a solid support may simply be a piece offilter membrane, such as one made of nitrocellulose or nylon. In thismanner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity forpolypeptides of the invention. Furthermore, the amino acid sequence ofthe polypeptide having a selective affinity for the polypeptides of theinvention can be determined directly by conventional means or the codingsequence of the DNA encoding the polypeptide can frequently bedetermined more conveniently. The primary sequence can then be deducedfrom the corresponding DNA sequence. If the amino acid sequence is to bedetermined from the polypeptide itself, one may use microsequencingtechniques. The sequencing technique may include mass spectroscopy.

In certain situations, it may be desirable to wash away any unboundpolypeptides from a mixture of the polypeptides of the invention and theplurality of polypeptides prior to attempting to determine or to detectthe presence of a selective affinity interaction. Such a wash step maybe particularly desirable when the polypeptides of the invention or theplurality of polypeptides are bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind polypeptides of the invention. Many libraries areknown in the art that can be used, e.g., chemically synthesizedlibraries, recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been pernethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CTPublication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindspolypeptides of the invention can be carried out by contacting thelibrary members with polypeptides of the invention immobilized on asolid phase and harvesting those library members that bind to thepolypeptides of the invention. Examples of such screening methods,termed “panning” techniques are described by way of example in Parmleyand Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques13:422-427; PCT Publication No. WO 94/18318; and in references citedherein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to polypeptides of theinvention.

Where the binding molecule is a polypeptide, the polypeptide can beconveniently selected from any peptide library, including random peptidelibraries, combinatorial peptide libraries, or biased peptide libraries.The term “biased” is used herein to mean that the method of generatingthe library is manipulated so as to restrict one or more parameters thatgovern the diversity of the resulting collection of molecules, in thiscase peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a binding polypeptide has in the range of 15-100 aminoacids, or 20-50 amino acids.

The selected binding polypeptide can be obtained by chemical synthesisor recombinant expression.

Other Activities

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention, as a result of the ability to stimulate vascular endothelialcell growth, may be employed in treatment for stimulatingre-vascularization of ischemic tissues due to various disease conditionssuch as thrombosis, arteriosclerosis, and other cardiovascularconditions. The polypeptide, polynucleotide, agonist, or antagonist ofthe present invention may also be employed to stimulate angiogenesis andlimb regeneration, as discussed above.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for treating wounds due to injuries,burns, post-operative tissue repair, and ulcers since they are mitogenicto various cells of different origins, such as fibroblast cells andskeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed stimulate neuronal growth and to treatand prevent neuronal damage which occurs in certain neuronal disordersor neuro-degenerative conditions such as Alzheimer's disease,Parkinson's disease, and AIDS-related complex. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may havethe ability to stimulate chondrocyte growth, therefore, they may beemployed to enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be also be employed to prevent skin aging due to sunburnby stimulating keratinocyte growth.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed for preventing hair loss, since FGFfamily members activate hair-forming cells and promotes melanocytegrowth. Along the same lines, a polypeptide, polynucleotide, agonist, orantagonist of the present invention may be employed to stimulate growthand differentiation of hematopoietic cells and bone marrow cells whenused in combination with other cytokines.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be employed to maintain organs before transplantationor for supporting cell culture of primary tissues. A polypeptide,polynucleotide, agonist, or antagonist of the present invention may alsobe employed for inducing tissue of mesodermal origin to differentiate inearly embryos.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used to modulate mammalian characteristics, suchas body height, weight, hair color, eye color, skin, percentage ofadipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, a polypeptide, polynucleotide, agonist, or antagonist of thepresent invention may be used to modulate mammalian metabolism affectingcatabolism, anabolism, processing, utilization, and storage of energy.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be used to change a mammal's mental state or physicalstate by influencing biorhythms, caricadic rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may also be used as a food additive or preservative, such asto increase or decrease storage capabilities, fat content, lipid,protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolatednucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a sequence of at least about 50 contiguous nucleotidesin the nucleotide sequence of SEQ ID NO:X or the complementary strandthereto, the nucleotide sequence as defined in column 5 of Table 1B.1 orcolumns 8 and 9 of Table 2 or the complementary strand thereto, and/orcDNA contained in ATCC Deposit No:Z.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of theportion of SEQ ID NO:X as defined in column 5, “ORF (From-To)”, in Table1B.1.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of theportion of SEQ ID NO:X as defined in columns 8 and 9, “NT From” and “NTTo” respectively, in Table 2.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X or the complementary strand thereto, the nucleotide sequence asdefined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or thecomplementary strand thereto, and/or cDNA contained in ATCC DepositNo:Z.

Further preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 500 contiguous nucleotides in the nucleotide sequence of SEQID NO:X or the complementary strand thereto, the nucleotide sequence asdefined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or thecomplementary strand thereto, and/or cDNA contained in ATCC DepositNo:Z.

A further preferred embodiment is a nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of the portion of SEQ ID NO:X defined in column 5, “ORF(From-To)”, in Table 1B.1.

A further preferred embodiment is a nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of the portion of SEQ ID NO:X defined in columns 8 and 9, “NTFrom” and “NT To”, respectively, in Table 2.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence of SEQ ID NO:X or the complementary strandthereto, the nucleotide sequence as defined in column 5 of Table 1B.1 orcolumns 8 and 9 of Table 2 or the complementary strand thereto, and/orcDNA contained in ATCC Deposit No:Z.

Also preferred is an isolated nucleic acid molecule which hybridizesunder stringent hybridization conditions to a nucleic acid moleculecomprising a nucleotide sequence of SEQ ID NO:X or the complementarystrand thereto, the nucleotide sequence as defined in column 5 of TablelB.1 or columns 8 and 9 of Table 2 or the complementary strand thereto,and/or cDNA contained in ATCC Deposit No:Z, wherein said nucleic acidmolecule which hybridizes does not hybridize under stringenthybridization conditions to a nucleic acid molecule having a nucleotidesequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA moleculewhich comprises the cDNA contained in ATCC Deposit No:Z.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides of the cDNA sequence contained in ATCCDeposit No:Z.

Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of an open reading frame sequence encoded by cDNAcontained in ATCC Deposit No:Z.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bycDNA contained in ATCC Deposit No:Z.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical tosequence of at least 500 contiguous nucleotides in the nucleotidesequence encoded by cDNA contained in ATCC Deposit No:Z.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence encoded by cDNA contained in ATCC DepositNo:Z.

A further preferred embodiment is a method for detecting in a biologicalsample a nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X or the complementary strand thereto;the nucleotide sequence as defined in column 5 of Table 1B.1 or columns8 and 9 of Table 2 or the complementary strand thereto; and a nucleotidesequence encoded by cDNA contained in ATCC Deposit No:Z; which methodcomprises a step of comparing a nucleotide sequence of at least onenucleic acid molecule in said sample with a sequence selected from saidgroup and determining whether the sequence of said nucleic acid moleculein said sample is at least 95% identical to said selected sequence.

Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

A further preferred embodiment is a method for identifying the species,tissue or cell type of a biological sample which method comprises a stepof detecting nucleic acid molecules in said sample, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X or the complementarystrand thereto; the nucleotide sequence as defined in column 5 of Table1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto;and a nucleotide sequence of the cDNA contained in ATCC Deposit No:Z.

The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of anucleotide sequence of SEQ ID NO:X or the complementary strand thereto;the nucleotide sequence as defined in column 5 of Table 1B.1 or columns8 and 9 of Table 2 or the complementary strand thereto; or the cDNAcontained in ATCC Deposit No:Z which encodes a protein, wherein themethod comprises a step of detecting in a biological sample obtainedfrom said subject nucleic acid molecules, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X or the complementarystrand thereto; the nucleotide sequence as defined in column 5 of Table1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto;and a nucleotide sequence of cDNA contained in ATCC Deposit No:Z.

The method for diagnosing a pathological condition can comprise a stepof detecting nucleic acid molecules comprising a nucleotide sequence ina panel of at least two nucleotide sequences, wherein at least onesequence in said panel is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:X orthe complementary strand thereto; the nucleotide sequence as defined incolumn 5 of Table 1B.1 or columns 8 and 9 of Table 2 or thecomplementary strand thereto; and a nucleotide sequence encoded by cDNAcontained in ATCC Deposit No:Z. The nucleic acid molecules can compriseDNA molecules or RNA molecules.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a DNA microarray or “chip” of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 500,1000, 2000, 3000, or 4000 nucleotide sequences, wherein at least onesequence in said DNA nicroarray or “chip” is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1A and/or 1B; and anucleotide sequence encoded by a human cDNA clone identified by a cDNA“Clone ID” in Table 1A and/or 1B.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand thereto;the polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained inATCC Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand thereto;the polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained inATCC Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand thereto;the polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained inATCC Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the complete amino acid sequence ofSEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementarystrand thereto; the polypeptide encoded by the nucleotide sequence asdefined in columns 8 and 9 of Table 2; and/or a polypeptide encoded bycDNA contained in ATCC Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of apolypeptide encoded by contained in ATCC Deposit No:Z

Also preferred is a polypeptide wherein said sequence of contiguousamino acids is included in the amino acid sequence of a portion of saidpolypeptide encoded by cDNA contained in ATCC Deposit No:Z; apolypeptide encoded by SEQ ID NO:X or the complementary strand thereto;the polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of a polypeptideencoded by the cDNA contained in ATCC Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of a polypeptideencoded by cDNA contained in ATCC Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the amino acid sequence of apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z.

Further preferred is an isolated antibody which binds specifically to apolypeptide comprising an amino acid sequence that is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: a polypeptide sequenceof SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or thecomplementary strand thereto; the polypeptide encoded by the nucleotidesequence as defined in columns 8 and 9 of Table 2; and a polypeptideencoded by the cDNA contained in ATCC Deposit No:Z.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: a polypeptide sequenceof SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or thecomplementary strand thereto; the polypeptide encoded by the nucleotidesequence as defined in columns 8 and 9 of Table 2; and a polypeptideencoded by the cDNA contained in ATCC Deposit No:Z; which methodcomprises a step of comparing an amino acid sequence of at least onepolypeptide molecule in said sample with a sequence selected from saidgroup and determining whether the sequence of said polypeptide moleculein said sample is at least 90% identical to said sequence of at least 10contiguous amino acids.

Also preferred is the above method wherein said step of comparing anamino acid sequence of at least one polypeptide molecule in said samplewith a sequence selected from said group comprises determining theextent of specific binding of polypeptides in said sample to an antibodywhich binds specifically to a polypeptide comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQID NO:X or the complementary strand thereto; the polypeptide encoded bythe nucleotide sequence as defined in columns 8 and 9 of Table 2; and apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z.

Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

Also preferred is a method for identifying the species, tissue or celltype of a biological sample which method comprises a step of detectingpolypeptide molecules in said sample, if any, comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ IDNO:X or the complementary strand thereto; the polypeptide encoded by thenucleotide sequence as defined in columns 8 and 9 of Table 2; and apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z.

Also preferred is the above method for identifying the species, tissueor cell type of a biological sample, which method comprises a step ofdetecting polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the abovegroup.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a nucleicacid sequence identified in Table 1A, 1B or Table 2 encoding apolypeptide, which method comprises a step of detecting in a biologicalsample obtained from said subject polypeptide molecules comprising anamino acid sequence in a panel of at least two amino acid sequences,wherein at least one sequence in said panel is at least 90% identical toa sequence of at least 10 contiguous amino acids in a sequence selectedfrom the group consisting of: polypeptide sequence of SEQ ID NO:Y; apolypeptide encoded by SEQ ID NO:X or the complementary strand thereto;the polypeptide encoded by the nucleotide sequence as defined in columns8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained inATCC Deposit No:Z.

In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto; thepolypeptide encoded by the nucleotide sequence as defined in columns 8and 9 of Table 2; and a polypeptide encoded by the cDNA contained inATCC Deposit No:Z.

Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

Also preferred is a polypeptide molecule, wherein said polypeptidecomprises an amino acid sequence selected from the group consisting of:polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ IDNO:X or the complementary strand thereto; the polypeptide encoded by thenucleotide sequence as defined in columns 8 and 9 of Table 2; and apolypeptide encoded by the cDNA contained in ATCC Deposit No:Z.

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecule into a vector.Also preferred is the recombinant vector produced by this method. Alsopreferred is a method of making a recombinant host cell comprisingintroducing the vector into a host cell, as well as the recombinant hostcell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is ahuman protein comprising an amino acid sequence selected from the groupconsisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptideencoded by SEQ ID NO:X or the complementary strand thereto; thepolypeptide encoded by the nucleotide sequence as defined in columns 8and 9 of Table 2; and a polypeptide encoded by the cDNA contained inATCC Deposit No:Z. The isolated polypeptide produced by this method isalso preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a Therapeutic comprising an amountof an isolated polypeptide, polynucleotide, immunogenic fragment oranalogue thereof, binding agent, antibody, or antigen binding fragmentof the claimed invention effective to increase the level of said proteinactivity in said individual.

Also preferred is a method of treatment of an individual in need of adecreased level of a protein activity, which method comprisedadministering to such an individual a Therapeutic comprising an amountof an isolated polypeptide, polynucleotide, immunogenic fragment oranalogue thereof, binding agent, antibody, or antigen binding fragmentof the claimed invention effective to decrease the level of said proteinactivity in said individual.

Also preferred is a method of treatment of an individual in need of aspecific delivery of toxic compositions to diseased cells (e.g., tumors,leukemias or lymphomas), which method comprises administering to such anindividual a Therapeutic comprising an amount of an isolated polypeptideof the invention, including, but not limited to a binding agent, orantibody of the claimed invention that are associated with toxin orcytotoxic prodrugs.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Description of Table 6

Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCCdesignation numbers of deposits made with the ATCC in connection withthe present application. These deposits were made in addition to thosedescribed in the Table 1A. TABLE 6 ATCC Deposits Deposit Date ATCCDesignation Number LP01, LP02, LP03, LP04, May 20, 1997 209059, 209060,209061, LP05, LP06, LP07, LP08, 209062, 209063, 209064, LP09, LP10,LP11, 209065, 209066, 209067, 209068, 209069 LP12 Jan. 12, 1998 209579LP13 Jan. 12, 1998 209578 LP14 Jul. 16, 1998 203067 LP15 Jul. 16, 1998203068 LP16 Feb. 1, 1999 203609 LP17 Feb. 1, 1999 203610 LP20 Nov. 17,1998 203485 LP21 Jun. 18, 1999 PTA-252 LP22 Jun. 18, 1999 PTA-253 LP23Dec. 22, 1999 PTA-1081

EXAMPLES Example 1 Isolation of a Selected cDNA Clone From the DepositedSample

Each ATCC Deposit No:Z is contained in a plasmid vector. Table 7identifies the vectors used to construct the cDNA library from whicheach clone was isolated. In many cases, the vector used to construct thelibrary is a phage vector from which a plasmid has been excised. Thefollowing correlates the related plasmid for each phage vector used inconstructing the cDNA library. For example, where a particular clone isidentified in Table 7 as being isolated in the vector “Lambda Zap,” thecorresponding deposited clone is in “pBluescript.” Vector Used toCorresponding Construct Library Deposited Plasmid Lambda Zap pBluescript(pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBK lafmid BA plafmid BApSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0pCR ® 2.1 pCR ® 2.1

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:5861 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Both can be transformed into E.coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of thepolylinker to the T7 and T3 primer sequences which flank the polylinkerregion (“S” is for Sacl and “K” is for KpnI which are the first sites oneach respective end of the linker). “+” or “−” refer to the orientationof the f1 origin of replication (“ori”), such that in one orientation,single stranded rescue initiated from the f1 ori generates sense strandDNA and in the other, antisense.

Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained fromLife Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. AllSport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993)). Vector lafmid BA (Bento Soares, Columbia University, N.Y.)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: 1991)). Preferably, a polynucleotide of the presentinvention does not comprise the phage vector sequences identified forthe particular clone in Table 7, as well as the corresponding plasmidvector sequences designated above.

The deposited material in the sample assigned the ATCC Deposit Numbercited by reference to Table 1A, Table 2, Table 6 and Table 7 for anygiven cDNA clone also may contain one or more additional plasmids, eachcomprising a cDNA clone different from that given clone. Thus, depositssharing the same ATCC Deposit Number contain at least a plasmid for eachATCC Deposit No:Z. TABLE 7 ATCC Libraries owned by Catalog CatalogDescription Vector Deposit HUKA HUKB HUKC HUKD Human Uterine CancerLambda ZAP II LP01 HUKE HUKF HUKG HCNA HCNB Human Colon Lambda Zap IILP01 HFFA Human Fetal Brain, Lambda Zap II LP01 random primed HTWAResting T-Cell Lambda ZAP II LP01 HBQA Early Stage Human Lambda ZAP IILP01 Brain, random primed HLMB HLMF HLMG HLMH breast lymph node LambdaZAP II LP01 HLMI HLMJ HLMM HLMN CDNA library HCQA HCQB human coloncancer Lamda ZAP II LP01 HMEA HMEC HMED HMEE Human Microvascular LambdaZAP II LP01 HMEF HMEG HMEI HMEJ Endothelial Cells, HMEK HMEL fract. AHUSA HUSC Human Umbilical Vein Lambda ZAP II LP01 Endothelial Cells,fract. A HLQA HLQB Hepatocellular Tumor Lambda ZAP II LP01 HHGA HHGBHHGC HHGD Hemangiopericytoma Lambda ZAP II LP01 HSDM Human StriatumDepression, Lambda ZAP II LP01 re-rescue HUSH H Umbilical Vein LambdaZAP II LP01 Endothelial Cells, frac A, re-excision HSGS Salivary gland,subtracted Lambda ZAP II LP01 HFXA HFXB HFXC HFXD Brain frontal cortexLambda ZAP II LP01 HFXE HFXF HFXG HFXH HPQA HPQB HPQC PERM TF274 LambdaZAP II LP01 HFXJ HFXK Brain Frontal Cortex, Lambda ZAP II LP01re-excision HCWA HCWB HCWC CD34 positive cells ZAP Express LP02 HCWDHCWE HCWF (Cord Blood) HCWG HCWH HCWI HCWJ HCWK HCUA HCUB HCUC CD34depleted Buffy ZAP Express LP02 Coat (Cord Blood) HRSM A-14 cell lineZAP Express LP02 HRSA A1-CELL LINE ZAP Express LP02 HCUD HCUE HCUF HCUGCD34 depleted Buffy ZAP Express LP02 HCUH HCUI Coat (Cord Blood),re-excision HBXE HBXF HBXG H. Whole Brain #2, ZAP Express LP02re-excision HRLM L8 cell line ZAP Express LP02 HBXA HBXB HBXC HBXD HumanWhole Brain #2 - ZAP Express LP02 Oligo dT > 1.5 Kb HUDA HUDB HUDCTestes ZAP Express LP02 HHTM HHTN HHTO H. hypothalamus, frac A; ZAPExpress LP02 re-excision HHTL H. hypothalamus, frac A ZAP Express LP02HASA HASD Human Adult Spleen Uni-ZAP XR LP03 HFKC HFKD HEKE HFKF HumanFetal Kidney Uni-ZAP XR LP03 HFKG HE8A HE8B HE8C HE8D Human 8 Week WholeUni-ZAP XR LP03 HE8E HE8F HE8M HE8N Embryo HGBA HGBD HGBE HGBF HumanGall Bladder Uni-ZAP XR LP03 HGBG HGBH HGBI HLHA HLHB HLHC HLHD HumanFetal Lung III Uni-ZAP XR LP03 HLHE HLHF HLHG HLHH HLHQ HPMA HPMB HPMCHPMD Human Placenta Uni-ZAP XR LP03 HPME HPMF HPMG HPMH HPRA HPRB HPRCHPRD Human Prostate Uni-ZAP XR LP03 HSIA HSIC HSID HSIE Human AdultSmall Uni-ZAP XR LP03 Intestine HTEA HTEB HTEC HTED Human Testes Uni-ZAPXR LP03 HTEE HTEF HTEG HTEH UTEI HTEJ HTEK HTPA HTPB HTPC HTPD HumanPancreas Tumor Uni-ZAP XR LP03 HTPE HTTA HTTB HTTC HTTD Human TestesTumor Uni-ZAP XR LP03 HTTE HTTF HAPA HAPB HAPC HAPM Human AdultPulmonary Uni-ZAP XR LP03 HETA HETB HETC HETD Human Endometrial TumorUni-ZAP XR LP03 HETE HETF HETG HETH HETI HHFB HHFC HHFD HHFE Human FetalHeart Uni-ZAP XR LP03 HHFF HHFG HHFH HHFI HHPB HHPC HHPD HHPE HumanHippocampus Uni-ZAP XR LP03 HHPF HHPG HHPH HCE1 HCE2 HCE3 HCE4 HumanCerebellum Uni-ZAP XR LP03 HCE5 HCEB HCEC HCED HCEE HCEF HCEG HUVB HUVCHUVD HUVE Human Umbilical Vein, Uni-ZAP XR LP03 Endo. remake HSTA HSTBHSTC HSTD Human Skin Tumor Uni-ZAP XR LP03 HTAA HTAB HTAC HTAD HumanActivated T-Cells Uni-ZAP XR LP03 HTAE HFEA HEEB HFEC Human FetalEpithelium Uni-ZAP XR LP03 (Skin) HJPA HJPB HJPC HJPD HUMAN JURKATUni-ZAP XR LP03 MEMBRANE BOUND POLYSOMES HESA Human epithelioid sarcomaUni-Zap XR LP03 HLTA HLTB HLTC HLTD Human T-Cell Lymphoma Uni-ZAP XRLP03 HLTE HLTF HFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XRLP03 HRDA HRDB HRDC HRDD Human Rhabdomyosarcoma Uni-ZAP XR LP03 HRDEHRDF HCAA HCAB HCAC Cem cells cyclohexamide Uni-ZAP XR LP03 treated HRGAHRGB HRGC HRGD Raji Cells, cyclohexamide Uni-ZAP XR LP03 treated HSUAHSUB HSUC HSUM Supt Cells, cyclohexamide Uni-ZAP XR LP03 treated HT4AHT4C HT4D Activated T-Cells, 12 hrs. Uni-ZAP XR LP03 HE9A HE9B HE9C HE9DNine Week Old Early Stage Uni-ZAP XR LP03 HE9E HE9F HE9G HE9H Human HE9MHE9N HATA HATB HATC HATD Human Adrenal Gland Tumor Uni-ZAP XR LP03 HATEHT5A Activated T-Cells, 24 hrs. Uni-ZAP XR LP03 HFGA HFGM Human FetalBrain Uni-ZAP XR LP03 HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XRLP03 HNEE HBGB HBGD Human Primary Breast Uni-ZAP XR LP03 Cancer HBNAHBNB Human Normal Breast Uni-ZAP XR LP03 HCAS Cem Cells, cyclohexamideUni-ZAP XR LP03 treated, subtra HHPS Human Hippocampus, pBS LP03subtracted HKCS HKCU Human Colon Cancer, pBS LP03 subtracted HRGS Rajicells, cyclohexamide pBS LP03 treated, subtracted HSUT Supt cells,cyclohexamide pBS LP03 treated, differentially expressed HT4S ActivatedT-Cells, 12 hrs, Uni-ZAP XR LP03 subtracted HCDA HCDB HCDC HCDD HumanChondrosarcoma Uni-ZAP XR LP03 HCDE HOAA HOAB HOAC Human OsteosarcomaUni-ZAP XR LP03 HTLA HTLB HTLC HTLD Human adult testis, Uni-ZAP XR LP03HTLE HTLF large inserts HLMA HLMC HLMD Breast Lymph node cDNA Uni-ZAP XRLP03 library H6EA H6EB H6EC HL-60, PMA 4H Uni-ZAP XR LP03 HTXA HTXB HTXCHTXD Activated T-Cell Uni-ZAP XR LP03 HTXE HTXF HTXG HTXH(12hs)/Thiouridine labelledEco HNFA HNFB HNFC HNFD Human Neutrophil,Uni-ZAP XR LP03 HNFE HNFF HNFG HNFH Activated HNFJ HTOB HTOC HUMANTONSILS, Uni-ZAP XR LP03 FRACTION 2 HMGB Human OB MG63 control Uni-ZAPXR LP03 fraction I HOPB Human OB HOS control Uni-ZAP XR LP03 fractionHORB Human OB HOS treated Uni-ZAP XR LP03 (10 nM E2) fraction I HSVAHSVB HSVC Human Chronic Synovitis Uni-ZAP XR LP03 HROA HUMAN STOMACHUni-ZAP XR LP03 HBJA HBJB HBJC HBJD HUMAN B CELL LYMPHOMA Uni-ZAP XRLP03 HBJE HBJF HBJG HBJH HBJI HBJJ HBJK HCRA HCRB HCRC human corpuscolosum Uni-ZAP XR LP03 HODA HODB HODC HODD human ovarian cancer Uni-ZAPXR LP03 HDSA Dermatofibrosarcoma Uni-ZAP XR LP03 Protuberance HMWA HMWBHMWC Bone Marrow Cell Line Uni-ZAP XR LP03 HMWD HMWE HMWF (Rs4;11) HMWGHMWH HMWI HMWJ HSOA stomach cancer (human) Uni-ZAP XR LP03 HERA SKINUni-ZAP XR LP03 HMDA Brain-medulloblastoma Uni-ZAP XR LP03 UGLA HGLBHGLD Glioblastoma Uni-ZAP XR LP03 HEAA H. Atrophic Endometrium Uni-ZAPXR LP03 HBCA HBCB H. Lymph node breast Cancer Uni-ZAP XR LP03 HPWT HumanProstate BPH, Uni-ZAP XR LP03 re-excision HFVG HFVH HFVI Fetal Liver,subtraction II pBS LP03 HNFI Human Neutrophils, pBS LP03 Activated,re-excision HBMB HBMC HBMD Human Bone Marrow, re- pBS LP03 excision HKMLHKMM HKMN H. Kidney Medulla, re- pBS LP03 excision HKIX HKIY H. KidneyCortex, subtracted pBS LP03 HADT H. Amygdala Depression, pBS LP03subtracted H6AS H1-60, untreated, Uni-ZAP XR LP03 subtracted H6ES HL-60,PMA 4H, Uni-ZAP XR LP03 subtracted H6BS HL-60, RA 4h, Subtracted Uni-ZAPXR LP03 H6CS HL-60, PMA 1d, subtracted Uni-ZAP XR LP03 HTXJ HTXKActivated T- Uni-ZAP XR LP03 cell(12h)/Thiouridine- re-excision HMSAHMSB HMSC HMSD Monocyte activated Uni-ZAP XR LP03 HMSE HMSF HMSG HMSHHMSI HMSJ HMSK HAGA HAGB HAGC HAGD Human Amygdala UnI-ZAP XR LP03 HAGEHAGF HSRA HSRB HSRE STROMAL- Uni-ZAP XR LP03 OSTEOCLASTOMA HSRD HSRFHSRG HSRH Human Osteoclastoma Uni-ZAP XR LP03 Stromal Cells-unamplifiedHSQA HSQB HSQC HSQD StroMal cell TF274 Uni-ZAP XR LP03 HSQE HSQF HSQGHSKA HSKB HSKC HSKD Smooth muscle, serum Uni-ZAP XR LP03 HSKE HSKF HSKZtreated HSLA HSLB HSLC HSLD Smooth muscle,control Uni-ZAP XR LP03 HSLEHSLF HSLG HSDA HSDD HSDE HSDF Spinal cord Uni-ZAP XR LP03 HSDG HSDH HPWSProstate-BPH subtracted II pBS LP03 HSKW HSKX HSKY Smooth Muscle- HASTEpBS LP03 normalized HFPB HFPC HFPD H. Frontal cortex,epileptic; Uni-ZALPXR LP03 re-excision HSDI HSDJ HSDK Spinal Cord, re-excision Uni-ZAP XRLP03 HSKN HSKO Smooth Muscle Serum pBS LP03 Treated, Norm HSKG HSKH HSKISmooth muscle, serum pBS LP03 induced,re-exc HFCA HFCB HFCC HFCD HumanFetal Brain Uni-ZAP XR LP04 HFCE HFCF HPTA HPTB HPTD Human PituitaryUni-ZAP XR LP04 HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP04 HB6B HE6CHE6D HE6E Human Whole Six Week Old Uni-ZAP XR LP04 HE6F HE6G HE6S EmbryoHSSA HSSB HSSC HSSD Human Synovial Sarcoma Uni-ZAP XR LP04 HSSE HSSFHSSG HSSH HSSI HSSJ HSSK HE7T 7 Week Old Early Stage Uni-ZAP XR LP04Human, subtracted HEPA HEPB HEPC Human Epididymus Uni-ZAF XR LP04 HSNAHSNB HSNC HSNM Human Synovium Uni-ZAP XR LP04 HSNN HPFB HPFC HPFD HPFEHuman Prostate Cancer, Uni-ZAP XR LP04 Stage C fraction HE2A HE2D HE2EHE2H 12 Week Old Early Stage Uni-ZAP XR LP04 HE2I HE2M HE2N HE2O HumanHE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP04 HE2P HE2QHuman, II HPTS HPTT HPTU Human Pituitary, subtracted Uni-ZAP XR LP04HAUA HAUB HAUC Amniotic Cells-TNF induced Uni-ZAP XR LP04 HAQA HAQB HAQCHAQD Amniotic Cells-Primary Culture Uni-ZAP XR LP04 HWTA HWTB HWTCwilm's tumor Uni-ZAP XR LP04 HBSD Bone Cancer, re-excision Uni-ZAP XRLP04 HSGB Salivary gland, re-excision Uni-ZAP XR LP04 HSJA HSJB HSJCSmooth muscle-ILb induced Uni-ZAP XR LP04 HSXA HSXB HSXC HSXD HumanSubstantia Nigra Uni-ZAP XR LP04 HSHA HSHB HSHC Smooth muscle, ILbinduced Uni-ZAP XR LP04 HOUA HOUB HOUC HOUD Adipocytes Uni-ZAP XR LP04HOUE HPWA HPWB HPWC HPWD Prostate BPH Uni-ZAP XR LP04 HPWE HELA HELBHELC HELD Endothelial cells-control Uni-ZAP XR LP04 HELE HELF HELG HELHHEMA HEMB HEMC HEMD Endothelial-induced Uni-ZAP XR LP04 HEME HEMF HEMGHEMH HBIA HBIB HBIC Human Brain, Striatum Uni-ZAP XR LP04 HHSA HHSB HHSCHHSD Human Uni-ZAP XR LP04 HHSE Hypothalmus, Schizophrenia HNGA HNGBHNGC HNGD neutrophils control Uni-ZAP XR LP04 HNGE HNGF HNGG HNGH HNGIHNGJ HNHA HNHB HNHC HNHD Neutrophils IL-1 and LPS Uni-ZAP XR LP04 HNHEHNHF HNHG HNHH induced HNHI HNHJ HSDB HSDC STRIATUM DEPRESSION Uni-ZAPXR LP04 HHPT Hypothalamus Uni-ZAP XR LP04 HSAT HSAU HSAV HSAW AnergicT-cell Uni-ZAP XR LP04 HSAX HSAY HSAZ HEMS HBMT HBMU HBMV Bone marrowUni-ZAP XR LP04 HBMW HBMX HOEA HOEB HOEC HOED Osteoblasts Uni-ZAP XRLP04 HOEE HOEF HOEJ HAIA HAIB HAIC HAID Epithelial-TNFa and INF Uni-ZAPXR LP04 HAIE HAIF induced HTGA HTGB HTGC HTGD Apoptotic T-cell Uni-ZAPXR LP04 HMCA HMCB HMCC Macrophage-oxLDL Uni-ZAP XR LP04 HMCD HMCE HMAAHMAB HMAC Macrophage (GM-CSF Uni-ZAP XR LP04 HMAD HMAE HMAF treated)HMAG HPHA Normal Prostate Uni-ZAP XR LP04 HPIA HPIB HPIC LNCAP prostatecell line Uni-ZAP XR LP04 HPJA HPJB HPJC PC3 Prostate cell line Uni-ZAPXR LP04 HOSE HOSF HOSG Human Osteoclastoma, re- Uni-ZAP XR LP04 excisionHTGE HTGF Apoptotic T-cell, Uni-ZAP XR LP04 re-excision HMAJ HMAK HMacrophage (GM-CSF Uni-ZAP XR LP04 treated), re-excision HACB HACC HACDHuman Adipose Tissue, Uni-ZAP XR LP04 re-excision HEPA H. FrontalCortex, Uni-ZAP XR LP04 Epileptic HFAA HFAB HFAC HFAD Alzheimer's,spongy Uni-ZAP XR LP04 HFAE change HFAM Frontal Lobe, Dementia Uni-ZAPXR LP04 HMIA HMIB HMIC Human Manic Depression Uni-ZAP XR LP04 TissueHTSA HTSE HTSF HTSG Human Thymus pBS LP05 HTSH HPBA HPBB HPBC HPBD HumanPineal Gland pBS LP05 HPBE HSAA HSAB HSAC HSA 172 Cells pBS LP05 HSBAHSBB HSBC HSBM HSC172 cells pBS LP05 HJAA HJAB HJAC HJAD Jurkat T-cellG1 phase pBS LP05 HJBA HJBB HJBC HJBD Jurkat T-Cell, S phase pBS LP05HAFA HAFB Aorta endothelial pBS LP05 cells +TNF-a HAWA HAWB HAWC HumanWhite Adipose pBS LP05 HTNA HTNB Human Thyroid pBS LP05 HONA NormalOvary, Premenopausal pBS LP05 HARA HARB Human Adult Retina pBS LP05 HLJAHLJB Human Lung pCMVSport 1 LP06 HOFM HOFN HOFO H. Ovarian Tumor, II,pCMVSport 2.0 LP07 OV5232 HOGA HOGB HOGC OV 10-3-95 pCMVSport 2.0 LP07HCGL CD34+cells, II pCMVSport 2.0 LP07 HDLA Hodgkin's Lymphoma IpCMVSport 2.0 LP07 HDTA HDTB HDTC HDTD Hodgkin's Lymphoma II pCMVSport2.0 LP07 HDTE HKAA HKAB HXAC HXAD Keratinocyte pCMVSport 2.0 LP07 HKAEHKAF HKAG HKAH HCIM CAPFINDER, Crohn's pCMVSport 2.0 LP07 Disease, lib 2HKAL Keratinocyte, lib 2 pCMVSport 2.0 LP07 HKAT Keratinocyte, lib 3pCMVSport 2.0 LP07 HNDA Nasal polyps pCMVSport 2.0 LP07 HDRA H. PrimaryDendritic pCMVSport 2.0 LP07 Cells, lib 3 HOHA HOHB HOHC HumanOsteoblasts II pCMVSport 2.0 LP07 HLDA HLDB HLDC Liver, HepatomapCMVSport 3.0 LP08 HLDN HLDO HLDP Human Liver, normal pCMVSport 3.0 LP08HMTA pBMC stimulated w/poly pCMVSport 3.0 LP08 I/C HNTA NTERA2, controlpCMVSport 3.0 LP08 HDPA HDPB HDPC HDPD Primary Dendritic Cells,pCMVSport 3.0 LP08 HDPF HDPG HDPH HDPI lib 1 HDPJ HDPK HDPM HDPN HDPOHDPP Primary Dendritic cells, pCMVSport 3.0 LP08 frac 2 HMUA HMUB HMUCMyoloid Progenitor Cell Line pCMVSport 3.0 LP08 HHEA HHEB HHEC HHED TCell helper I pCMVSport 3.0 LP08 HHEM HHEN HHEO HHEP T cell helper IIpCMVSport 3.0 LP08 HEQA HEQB HEQC Human endometrial stromal pCMVSport3.0 LP08 cells HJMA HJMB Human endometrial stromal pCMVSport 3.0 LP08cells-treated with progesterone HSWA HSWB HSWC Human endometrial stromalpCMVSport 3.0 LP08 cells-treated with estradiol HSYA HSYB HSYC HumanThymus Stromal Cells pCMVSport 3.0 LP08 HLWA HLWB HLWC Human PlacentapCMVSport 3.0 LP08 HRAA HRAB HRAC Rejected Kidney, lib 4 pCMVSport 3.0LP08 HMTM PCR, pBMC I/C treated PCRII LP09 HMJA H. Meniingima, M6 pSport1 LP10 HMKA HMKB HMKC H. Meningima, M1 pSport 1 LP10 HMKD HMKE HUSG HUSIHuman umbilical vein pSport 1 LP10 endothelial cells, IL-4 induced HUSXHUSY Human Umbilical Vein pSport 1 LP10 Endothelial Cells, uninducedHOFA Ovarian Tumor I, OV5232 pSport 1 LP10 HCFA HCFB HCFC HCFD T-CellPHA 16 hrs pSport 1 LP10 HCFL HCFM HCFN HCFO T-Cell PHA 24 hrs pSport 1LP10 HADA HADC HADD HADE Human Adipose pSport 1 LP10 HADF HADG HOVA HOVBHOVC Human Ovary pSport 1 LP10 HTWB HTWC HTWD HTWE Resting T-CellLibrary, II pSport 1 LP10 HTWF HMMA Spleen metastic melanoma pSport 1LP10 HLYA HLYB HLYC HLYD Spleen, Chronic lymphocytic pSport 1 LP10 HLYEleukemia HCGA CD34+ cell, I pSport 1 LP10 HEOM HEON Human EosinophilspSport 1 LP10 HTDA Human Tonsil, Lib 3 pSport 1 LP10 HSPA SalivaryGland, Lib 2 pSport 1 LP10 HCHA HCHB HCHC Breast Cancer cell line,pSport 1 LP10 MDA 36 HCHM HCHN Breast Cancer Cell line, pSport 1 LP10angiogenic HCIA Crohn's Disease pSport 1 LP10 HDAA HDAB HDAC HEL cellline pSport 1 LP10 HABA Human Astrocyte pSport 1 LP10 HUFA HUFB HUFCUlcerative Colitis pSport 1 LP10 HNTM NTERA2 + retinoic acid, pSport 1LP10 14 days HDQA Primary Dendritic pSport 1 LP10 cells, CapFinder2,frac 1 HDQM Primary Dendritic Cells, pSport 1 LP10 CapFinder, frac 2HLDX Human Liver, normal, pSport 1 LP10 CapFinder HULA HULB HULC HumanDermal Endothelial pSport 1 LP10 Cells, untreated HUMA Human DermalEndothelial pSport 1 LP10 cells, treated HCJA Human Stromal EndometrialpSport 1 LP10 fibroblasts, untreated HCJM Human Stromal endometrialpSport 1 LP10 fibroblasts, treated w/estradiol HEDA Human Stromalendometrial pSport 1 LP10 fibroblasts, treated with progesterone HFNAHuman ovary tumor cell pSport 1 LP10 OV350721 HKGA HKGB HXGC HKGD MerkelCells pSport 1 LP10 HISA HISB HISC Pancreas Islet Cell Tumor pSport 1LP10 HLSA Skin, burned pSport 1 LP10 HBZA Prostate, BPH, Lib 2 pSport 1LP10 HBZS Prostate BPH, Lib 2, subtracted PSport 1 LP10 HFIA HFIB HFICSynovial Fibroblasts (control) pSport 1 LP10 HFIH HFII HFIJ Synovialhypoxia pSport 1 LP10 HFIT HFIU HFIV Synovial IL-1/TNF stimulated pSport1 LP10 HGCA Messangial cell, frac 1 pSport 1 LP10 HMVA HMVB HMVC BoneMarrow Stromal Cell, pSport 1 LP10 untreated HFIX HFIY HFIZ SynovialFibroblasts pSport 1 LP10 (II1/TNF), subt HFOX HFOY HFOZ Synovialhypoxia-RSF pSport 1 LP10 subtracted HMQA HMQB HMQC Human ActivatedMonocytes Uni-ZAP XR LP11 HMQD HLIA HLIB HLIC Human Liver pCMVSport 1LP012 HHBA HHBB HHBC HHBD Human Heart pCMVSport 1 LP012 HHBE HBBA HBBBHuman Brain pCMVSport 1 LP012 HLJA HLJB HLJG HLJD Human Lung pCMVSport 1LP012 HLJE HOGA HOGB HOGC Ovarian Tumor pCMVSport 2.0 LP012 HTJM HumanTonsils, Lib 2 pCMVSport 2.0 LP012 HAMF HAMG KMH2 pCMVSport 3.0 LP012HAJA HAJB HAJC L428 pCMVSport 3.0 LP012 HWBA HWBB HWBC Dendritic cells,pCMVSport 3.0 LP012 HWBD HWBE pooled HWAA HWAB HWAC Human Bone Marrow,pCMVSport 3.0 LP012 HWAD HWAE treated HYAA HYAB HYAC B Cell lymphomapCMVSport 3.0 LP012 HWHG HWHH HWHI Healing groin wound, pCMVSport 3.0LP012 6.5 hours post incision HWHP HWHQ HWHR Healing groin wound;pCMVSport 3.0 LP012 7.5 hours post incision HARM Healing groin wound-pCMVSport 3.0 LP012 zero hr post-incision (control) HBIM Olfactoryepithelium; pCMVSport 3.0 LP012 nasalcavity HWDA Healing Abdomen wound;pCMVSport 3.0 LP012 70&90 min post incision HWEA Healing AbdomenWound;15 pCMVSport 3.0 LP012 days post incision HWJA Healing AbdomenWound; pCMVSport 3.0 LP012 21&29 days HNAL Human Tongue, frac 2 pSport 1LP012 HMJA H. Meniingima, M6 pSport 1 LP012 HMKA HMKB HMKC H. Meningima,M1 pSport 1 LP012 HMKD HMKE HOFA Ovarian Tumor I, OV5232 pSport 1 LP012HCFA HCFB HCFC HCFD T-Cell PHA 16 hrs pSport 1 LP012 HCFL HCFM HCFN HCFOT-Cell PHA 24 hrs pSport 1 LP012 HMMA HMMB HMMC Spleen metastic melanomapSport 1 LP012 HTDA Human Tonsil, Lib 3 pSport 1 LP012 HDBA Human FetalThymus pSport 1 LP012 HDUA Pericardium pSport 1 LP012 HBZA Prostate,BPH,Lib 2 pSport 1 LP012 HWCA Larynx tumor pSport 1 LP012 HWKA Normal lungpSport 1 LP012 HSMB Bone marrow stroma, pSport 1 LP012 treated HBHMNormal trachea pSport 1 LP012 HLFC Human Larynx pSport 1 LP012 HLRBSiebben Polyposis pSport 1 LP012 HNIA Mammary Gland pSport 1 LP012 HNJBPalate carcinoma pSport 1 LP012 HNKA Palate normal pSport 1 LP012 HMZAPharynx carcinoma pSport 1 LP012 HABG Cheek Carcinoma pSport 1 LP012HMZM Pharynx Carcinoma pSport 1 LP012 HDRM Larynx Carcinoma pSport 1LP012 HVAA Pancreas normal PCA4 No pSport 1 LP012 HICA Tongue carcinomapSport 1 LP012 HUKA HUKB HUKC HUKD Human Uterine Cancer Lambda ZAP IILP013 HUKE HFFA Human Fetal Brain, Lambda ZAP II LP013 random primedHTUA Activated T-ceIl labeled with 4- Lambda ZAP II LP013 thioluri HBQAEarly Stage Human Brain, Lambda ZAP II LP013 random primed HMEB Humanmicrovascular Lambda ZAP II LP013 Endothelial cells, fract. B HUSH HumanUmbilical Vein Lambda ZAP II LP013 Endothelial cells, fract. A,re-excision HLQC HLQD Hepatocellular tumor, Lambda ZAP II LP013re-excision HTWJ HTWK HTWL Resting T-ceIl, re-excision Lambda ZAP IILP013 HF6S Human Whole 6 week Old pBluescript LP013 Embryo (II), subtHHPS Human Hippocampus, pBluescript LP013 subtracted HL1S LNCAP,differential pBluescnpt LP013 expression HLHS HLHT Early Stage HumanLung, pBluescript LP013 Subtracted HSUS Supt cells, cyclohexamidepBluescript LP013 treated, subtracted HSUT Supt cells, cyclohexamidepBluescript LP013 treated, differentially expressed HSDS H. StriatumDepression, pBluescript LP013 subtracted HPTZ Human Pituitary,Subtracted Bluescript LP013 VII HSDX H. Striatum Depression, pBluescriptLP013 subt II HSDZ H. Striatum Depression, pBluescript LP013 subt HPBAHPBB HPBC HPBD Human Pineal Gland pBluescript SK- LP013 HPBE HRTAColorectal Tumor pBluescript SK- LP013 HSBA HSBB HSBC HSBM H5C172 cellspBluescropt SK- LP013 HJAA HJAB HJAC HJAD Jurkat T-cell G1 phasepBluescript SK- LP013 HJBA HJBB HJBC HJBD Jurkat T-cell, S1 phasepBluescript SK- LP013 HTNA HTNB Human Thyroid pBluescript SK- LP013 HAHAHAHB Human Adult Heart Uni-ZAP XR LP013 HE6A Whole 6 week Old EmbryoUni-ZAP XR LP013 HFCA HFCB HFCC HFCD Human Fetal Brain Uni-ZAP XR LP013HFCE HFKC HFKD HFKE HFKF Human Fetal Kidney Uni-ZAP XR LP013 HFKG HGBAHGBD HGBE HGBF Human Gall Bladder Uni-ZAP XR LP013 HGBG HPRA HPRB HPRCHPRD Human Prostate Uni-ZAP XR LP013 HTEA HTEB HTEC HTED Human TestesUni-ZAP XR LP013 HTEE HTTA HTTB HTTC HTTD Human Testes Tumor Uni-ZAP XRLP013 HTTE HYBA HYBB Human Fetal Bone Uni-ZAP XR LP013 HFLA Human FetalLiver Uni-ZAP XR LP013 HHFB HHFC HHFD HHFE Human Fetal Heart Uni-ZAP XRLP013 HHFF HUVB HUVC HUVD HUVE Human Umbilical Vein, Uni-ZAP XR LP013End. remake HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP013 HSTA HSTB HSTCHSTD Human Skin Tumor Uni-ZAP XR LP013 HTAA HTAB HTAC HTAD HumanActivated T-cells Uni-ZAP XR LP013 HTAE HFEA HFEB HFEC Human FetalEpithelium Uni-ZAP XR LP013 (skin) HJPA HJPB HJPC HJPD Human JurkatMembrane Uni-ZAP XR LP013 Bound Polysomes HESA Human Epithelioid SarcomaUni-ZAF XR LP013 HALS Human Adult Liver, Uni-ZAP XR LP013 SubtractedHFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XR LP013 HCAA HCABHCAC Cem cells, cyclohexamide Uni-ZAP XR LP013 treated HRGA HRGB HRGCHRGD Raji Cells, cyclohexamide Uni-ZAP XR LP013 treated HE9A HE9B HE9CHE9D Nine Week Old Early Stage Uni-ZAP XR LP013 HE9E Human HSFA HumanFibrosarcoma Uni-ZAP XR LP013 HATA HATB HATC HATD Human Adrenal GlandUni-ZAP XR LP013 HATE Tumor HTRA Human Trachea Tumor Uni-ZAP XR LP013HE2A HE2D HE2E HE2H 12 Week Old Early Uni-ZAP XR LP013 HE2I Stage HumanHE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP013 HE2P Human,II HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XR LP013 HNEE HBGA HumanPrimary Uni-ZAP XR LP013 Breast Cancer HPTS HPTT HPTU Human Pituitary,Uni-ZAP XR LP013 subtracted HMQA HMQB HMQC Human Activated Uni-ZAP XRLP013 HMQD Monocytes HOAA HOAB HOAC Human Osteosarcoma Uni-ZAP XR LP013HTOA HTOD HTOE HTOF human tonsils Uni-ZAP XR LP013 HTOG HMGB Human OBMG63 control Uni-ZAP XR LP013 fraction I HOPB Human OB HOS controlUni-ZAP XR LP013 fraction HOQB Human OB HOS treated (1 nM Uni-ZAP XRLP013 E2) fraction I HAUA HAUB HAUC Amniotic Cells-TNF Uni-ZAP XR LP013induced HAQA HAQB HAQC HAQD Amniotic Cells-Primary Uni-ZAP XR LP013Culture HROA HROC HUMAN STOMACH Uni-ZAP XR LP013 HBJA HBJB HBJC HBJDHUMAN B CELL LYMPHOMA Uni-ZAP XR LP013 HBJE HODA HODB HODC HODD humanovarian cancer Uni-ZAP XR LP013 HCPA Corpus Callosum Urn-ZAP XR LP013HSOA stomach cancer (human) Uni-ZAP XR LP013 HERA SKIN Uni-ZAP XR LP013HMDA Brain-medulloblastoma Uni-ZAP XR LP013 HGLA HGLB HGLD GlioblastomaUni-ZAP XR LP013 HWTA HWTB HWTC wilm's tumor Urn-ZAP XR LP013 HEAA H.Atrophic Endometrium Uni-ZAP XR LP013 HAPN HAPO HAPP HAPQ Human AdultPulmonary; Uni-ZAP XR LP013 HAPR re-excision HLTG HLTH Human T-celllymphoma; Uni-ZAP XR LP013 re-excision HAHC HAHD HAHE Human Adult Heart;Uni-ZAP XR LP013 re-excision HAGA HAGB HAGC HAGD Human Amygdala Uni-ZAPXR LP013 HAGE HSJA HSJB HSJC Smooth muscle-ILb Uni-ZAP XR LP013 inducedHSHA HSHB HSHC Smooth muscle, IL1b Uni-ZAP XR LP013 induced HPWA HPWBHPWC HPWD Prostate BPH Uni-ZAP XR LP013 HPWE HPIA HPIB HPIC LNCAPprostate cell Uni-ZAP XR LP013 line HPJA HPJB HPJC PC3 Prostate cellline Uni-ZAP XR LP013 HBTA Bone Marrow Stroma, Uni-ZAP XR LP013 TNF&LPSind HMCF HMCG HMCH HMCI Macrophage-oxLDL; Uni-ZAP XR LP013 HMCJre-excision HAGG HAGH HAGI Human Amygdala;re-excision Uni-ZAP XR LP013HACA H. Adipose Tissue Uni-ZAP XR LP013 HKFB K562 + PMA (36 hrs), ZAPExpress LP013 re-excision HCWT HCWU HCWV CD34 positive cells (cord ZAPExpress LP013 blood),re-ex HBWA Whole brain ZAP Express LP013 HBXA HBXBHBXC HBXD Human Whole Brain #2- ZAP Express LP013 Oligo dT > 1.5 Kb HAVMTemporal cortex-Alzheizmer pT-Adv LP014 HAVT Hippocampus, AlzheimerpT-Adv LP014 Subtracted HHAS CHME Cell Line Uni-ZAP XR LP014 HAJR Larynxnormal pSport 1 LP014 HWLE HWLF HWLG HWLH Colon Normal pSport 1 LP014HCRM HCRN HCRO Colon Carcinoma pSport 1 LP014 HWLI HWLJ HWLK ColonNormal pSport 1 LP014 HWLQ HWLR HWLS HWLT Colon Tumor pSport 1 LP014HBFM Gastrocnemius Muscle pSport 1 LP014 HBOD HBOE Quadriceps MusclepSport 1 LP014 HBKD HBKE Soleus Muscle pSport 1 LP014 HCCM PancreaticLangerhans pSport 1 LP014 HWGA Larynx carcinoma pSport 1 LP014 HWGM HWGNLarynx carcinoma pSport 1 LP014 HWLA HWLB HWLC Normal colon pSport 1LP014 HWLM HWLN Colon Tumor pSport 1 LP014 HVAM HVAN HVAO Pancreas TumorpSport 1 LP014 HWGQ Larynx carcinoma nSport 1 LP014 HAQM HAQN SalivaryGland pSport 1 LP014 HASM Stomach; normal pSport 1 LP014 HBCM Uterus;normal pSport 1 LP014 HCDM Testis; normal pSport 1 LP014 HDJM Brain;normal pSport 1 LP014 HEFM Adrenal Gland, normal pSport 1 LP014 HBAARectum normal pSport 1 LP014 HFDM Rectum tumour pSport 1 LP014 HGAMColon, normal pSport 1 LP014 HHMM Colon, tumour pSport 1 LP014 HCLB HCLCHuman Lung Cancer Lambda Zap II LP015 HRLA L1 Cell line ZAP ExpressLP015 HHAM Hypothalamus, Alzheimer's pCMVSport 3.0 LP015 HKBA Ku 812FBasophils Line pSport 1 LP015 HS2S Saos2, Dexamethosome Treated pSport 1LP016 HA5A Lung Carcinoma A549 TNFalpha pSport 1 LP016 activated HTFMTF-1 Cell Line GM-CSF Treated pSport 1 LP016 HYAS Thyroid Tumour pSport1 LP016 HUTS Larynx Normal pSport 1 LP016 HXOA Larynx Tumor pSport 1LP016 HEAH Ea.hy.926 cell line pSport 1 LP016 HINA Adenocarcinoma HumanpSport 1 LP016 HRMA Lung Mesothelium pSport 1 LP016 HLCL HumanPre-Differentiated Uni-Zap XR LP017 Adipocytes HS2A Saos2 Cells pSport 1LP020 HS2I Saos2 Cells; Vitamin pSport 1 LP020 D3 Treated HUCM CHME CellLine, untreated pSport 1 LP020 HEPN Aryepiglottis Normal pSport 1 LP020HPSN Sinus Piniformis Tumour pSport 1 LP020 HNSA Stomach Normal pSport 1LP020 HNSM Stomach Tumour pSport 1 LP020 HNLA Liver Normal Met5No pSport1 LP020 HUTA Liver Tumour Met 5 Tu pSport 1 LP020 HOCN Colon NormalpSport 1 LP020 HOCT Colon Tumor pSport 1 LP020 HTNT Tongue Tumour pSport1 LP020 HLXN Larynx Normal pSport 1 LP020 HLXT Larynx Tumour pSport 1LP020 HTYN Thymus pSport 1 LP020 HPLN Placenta pSport 1 LP020 HTNGTongue Normal pSport 1 LP020 HZAA Thyroid Normal pSport 1 LP020 (SDCA2No) HWES Thyroid Thyroiditis pSport 1 LP020 HFHD Ficolled Human StromalpTrip1Ex2 LP021 Cells, 5Fu treated HFHM, HFHN Ficolled Human StromalpTrip1Ex2 LP021 Cells, Untreated HPCI Hep G2 Cells, lambda librarylambda Zap- LP021 CMV XR HBCA, HBCB, HBCC H. Lymph node breast CancerUni-ZAP XR LP021 HCOK Chondrocytes pSPORT1 LP022 HDCA, HDCB, HDCCDendritic Cells From pSPORT1 LP022 CD34 Cells HDMA, HDMB CD40 activatedmonocyte pSPORT1 LP022 dendritic cells HDDM, HDDN, UDDO LPS activatedderived pSPORT1 LP022 dendritic cells HPCR Hep G2 Cells, PCR librarylambda Zap- LP022 CMV XR HAAA, HAAB, HAAC Lung, Cancer (4005313A3):pSPORT1 LP022 Invasive Poorly Differentiated Lung Adenocarcinoma HIPA,HIPB, HIPC Lung, Cancer (4005163 B7): pSPORT1 LP022 Invasive, PoorlyDiff. Adenocarcinoma, Metastatic HOOH, HOOI Ovary, Cancer: (4004562 B6)pSPORT1 LP022 Papillary Serous Cystic Neoplasm, Low Malignant Pot HIDALung, Normal: (4005313 B1) pSPORT1 LP022 HUJA, HUJB, HUJC, B-CellspCMVSport 3.0 LP022 HUJD, HUJE HNOA, HNOB, HNOC, Ovary, Normal:(9805C040R) pSPORT1 LP022 HNOD HNLM Lung, Normal: (4005313 B1) pSPORT1LP022 HSCL Stromal Cells pSPORT1 LP022 HAAX Lung, Cancer: (4005313 A3)pSPORT1 LP022 Invasive Poorly-differentiated Metastatic lungadenocarcinoma HUUA, HUUB, HUUC, B-cells (unstimulated) pTrip1Ex2 LP022HUUD HWWA, HWWB, HWWC, B-cells (stimulated) pSPORT1 LP022 HWWD, HWWE,HWWF, HWWG HCCC Colon, Cancer: (9808C064R) pCMVSport 3.0 LP023 HPDO HPDPHPDQ HPDR Ovary, Cancer (9809C332): pSport 1 LP023 HPD Poorlydifferentiated adenocarcinoma HPCO HPCP HPCQ HPCT Ovary, Cancer(15395A1F): pSport 1 LP023 Grade II Papillary Carcinoma HOCM HOCO HOCPHOCQ Ovary, Cancer: (15799A1F) pSport 1 LP023 Poorly differentiatedcarcinoma HCBM HCBN HCBO Breast, Cancer: (4004943 A5) pSport 1 LP023HNBT HNBU HNBV Breast, Normal: (4005522B2) pSport 1 LP023 HBCP HBCQBreast, Cancer: (4005522 A2) pSport 1 LP023 HBCJ Breast, Cancer:(9806C012R) pSport 1 LP023 HSAM HSAN Stromal cells 3.88 pSport 1 LP023HVCA HVCB HVCC HVCD Ovary, Cancer: (4004332 A2) pSport 1 LP023 HSCK HSENHSEO Stromal cells (HBM3.18) pSport 1 LP023 HSCP HSCQ stromal cell clone2.5 pSport 1 LP023 HUXA Breast Cancer: (4005385 A2) pSport 1 LP023 HCOMHCON HCOO HCOP Ovary, Cancer (4004650 A3): pSport 1 LP023 HCOQWell-Differentiated Micropapillary Serous Carcinoma HBNM Breast, Cancer:(9802C020E) pSport 1 LP023 HVVA HVVB HVVC HVVD Human Bone Manow, treatedpSport 1 LP023 HVVE

Two nonlimiting examples are provided below for isolating a particularclone from the deposited sample of plasmid cDNAs cited for that clone inTable 7. First, a plasmid is directly isolated by screening the clonesusing a polynucleotide probe corresponding to the nucleotide sequence ofSEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the nucleotide sequence of SEQ ID NO:X are synthesized and used toamplify the desired cDNA using the deposited cDNA plasmid as a template.The polymerase chain reaction is carried out under routine conditions,for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v)gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C.for 1 min) are performed with a Perkin-Elmer Cetus automated thermalcycler. The amplified product is analyzed by agarose gel electrophoresisand the DNA band with expected molecular weight is excised and purified.The PCR product is verified to be the selected sequence by subcloningand sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of a gene which may not be present in the depositedclone. These methods include but are not limited to, filter probing,clone enrichment using specific probes, and protocols similar oridentical to 5′ and 3′ “RACE” protocols which are well known in the art.For instance, a method similar to 5′ RACE is available for generatingthe missing 5′ end of a desired full-length transcript. (Fromont-Racineet al., Nucleic Acids Res. 21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the sequence corresponding to SEQ ID NO:Xaccording to the method described in Example 1. (See also, Sambrook.)

Example 3 Tissue Specific Expression Analysis

The Human Genome Sciences, Inc. (HGS) database is derived fromsequencing tissue and/or disease specific cDNA libraries. Librariesgenerated from a particular tissue are selected and the specific tissueexpression pattern of EST groups or assembled contigs within theselibraries is determined by comparison of the expression patterns ofthose groups or contigs within the entire database. ESTs and assembledcontigs which show tissue specific expression are selected.

The original clone from which the specific EST sequence was generated,or in the case of an assembled contig, the clone from which the 5′ mostEST sequence was generated, is obtained from the catalogued library ofclones and the insert amplified by PCR using methods known in the art.The PCR product is denatured and then transferred in 96 or 384 wellformat to a nylon membrane (Schleicher and Scheull) generating an arrayfilter of tissue specific clones. Housekeeping genes, maize genes, andknown tissue specific genes are included on the filters. These targetscan be used in signal normalization and to validate assay sensitivity.Additional targets are included to monitor probe length and specificityof hybridization.

Radioactively labeled hybridization probes are generated by first strandcDNA synthesis per the manufacturer's instructions (Life Technologies)from mRNA/RNA samples prepared from the specific tissue being analyzed(e.g., prostate, prostate cancer, ovarian, ovarian cancer, etc.). Thehybridization probes are purified by gel exclusion chromatography,quantitated, and hybridized with the array filters in hybridizationbottles at 65° C. overnight. The filters are washed under stringentconditions and signals are captured using a Fuji phosphorimager.

Data is extracted using AIS software and following backgroundsubtraction, signal normalization is performed. This includes anormalization of filter-wide expression levels between differentexperimental runs. Genes that are differentially expressed in the tissueof interest are identified.

Example 4 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:X. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95° C.; 1 minute, 56°C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5minute cycle at 70° C. Human, mouse, and hamster DNA is used as templatein addition to a somatic cell hybrid panel containing individualchromosomes or chromosome fragments (Bios, Inc). The reactions areanalyzed on either 8% polyacrylamide gels or 3.5% agarose gels.Chromosome mapping is determined by the presence of an approximately 100bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/nl) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4° C. The cell debris is removed by centrifugation, and the supernatantcontaining the polypeptide is loaded onto a nickel-nitrilo-tri-aceticacid (“Ni—NTA”) affinity resin column (available from QIAGEN, Inc.,supra). Proteins with a 6×His tag bind to the Ni—NTA resin with highaffinity and can be purified in a simple one-step procedure (for detailssee: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni—NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

In addition to the above expression vector, the present inventionfurther includes an expression vector, called pHE4a (ATCC AccessionNumber 209645, deposited on Feb. 25, 1998) which contains phage operatorand promoter elements operatively linked to a polynucleotide of thepresent invention, called pHE4a. (ATCC Accession Number 209645,deposited on Feb. 25, 1998.) This vector contains: 1) aneomycinphosphotransferase gene as a selection marker, 2) an E. coliorigin of replication, 3) a T5 phage promoter sequence, 4) two lacoperator sequences, 5) a Shine-Delgarno sequence, and 6) the lactoseoperon repressor gene (lacIq). The origin of replication (oriC) isderived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operatorsequences are made synthetically.

DNA can be inserted into the pHE4a by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells harvested by continuouscentrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of theexpected yield of protein per unit weight of cell paste and the amountof purified protein required, an appropriate amount of cell paste, byweight, is suspended in a buffer solution containing 100 mM Tris, 50 mMEDTA, pH 7.4. The cells are dispersed to a homogeneous suspension usinga high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 MM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000 ×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4° C. overnight to allow furtherGuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 μm membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerseptiveBiosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pA2 is used to insert apolynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon, is amplified using the PCR protocoldescribed in Example 1. If a naturally occurring signal sequence is usedto produce the polypeptide of the present invention, the pA2 vector doesnot need a second signal peptide. Alternatively, the vector can bemodified (pA2 GP) to include a baculovirus leader sequence, using thestandard methods described in Summers et al., “A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five μg of a plasmid containing the polynucleotide is co-transfectedwith 1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA, Pharmingen, San Diego, Calif.), using thelipofection method described by Felgner et al., Proc. Natl. Acad. Sci.USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid are mixed in a sterile well of a microtiter plate containing 50μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg,Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added,mixed and incubated for 15 minutes at room temperature. Then thetransfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's mediumwithout serum. The plate is then incubated for 5 hours at 27° C. Thetransfection solution is then removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum is added.Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as DHFR, gpt, neomycin, orhygromycin allows the identification and isolation of the transfectedcells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991)). Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

A polynucleotide of the present invention is amplified according to theprotocol outlined in Example 1. If a naturally occurring signal sequenceis used to produce the polypeptide of the present invention, the vectordoes not need a second signal peptide. Alternatively, if a naturallyoccurring signal sequence is not used, the vector can be modified toinclude a heterologous signal sequence. (See, e.g., InternationalPublication No. WO 96/34891.) The amplified fragment is isolated from a1% agarose gel using a commercially available kit (“Geneclean,” BIO 101Inc., La Jolla, Calif.). The fragment then is digested with appropriaterestriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coli BB101 or XL-1 Blue cells are then transformed and bacteria are identifiedthat contain the fragment inserted into plasmid pC6 using, for instance,restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 μg of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 μM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reversedphase HPLC analysis.

Example 9 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988)). Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (ATCC Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide of the present invention, isolated by the PCR protocoldescribed in Example 1, is ligated into this BamHI site. Note that thepolynucleotide is cloned without a stop codon, otherwise a fusionprotein will not be produced.

If the naturally occurring signal sequence is used to produce thepolypeptide of the present invention, pC4 does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., International Publication No. WO 96/34891.) HumanIgG Fc region: (SEQ ID NO: 1)GGAATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing a polypeptide of the present invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of apolypeptide of the present invention is prepared and purified to renderit substantially free of natural contaminants. Such a preparation isthen introduced into an animal in order to produce polyclonal antiseraof greater specific activity.

Monoclonal antibodies specific for a polypeptide of the presentinvention are prepared using hybridoma technology (Kohler et al., Nature256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler etal., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and TCell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). Ingeneral, an animal (preferably a mouse) is immunized with a polypeptideof the present invention or, more preferably, with a secretedpolypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Eagle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide of thepresent invention.

Alternatively, additional antibodies capable of binding to a polypeptideof the present invention can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thepolypeptide-specific antibody can be blocked by said polypeptide. Suchantibodies comprise anti-idiotypic antibodies to thepolypeptide-specific antibody and are used to immunize an animal toinduce formation of further polypeptide-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., InternationalPublication No. WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985)).

b) Isolation Of Antibody Fragments Directed Against a Polypeptide of thePresent Invention From A Library Of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againsta polypeptide of the present invention to which the donor may or may nothave been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated hereinby reference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in International Publication No. WO 92/01047. Torescue phage displaying antibody fragments, approximately 109 E. coliharboring the phagemid are used to inoculate 50 ml of 2xTY containing 1%glucose and 100 μg/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D.of 0.8 with shaking. Five ml of this culture is used to inoculate 50 mlof 2xTY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene m, seeInternational Publication No. WO 92/01047) are added and the cultureincubated at 37° C. for 45 minutes without shaking and then at 37° C.for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.mfor 10 min. and the pellet resuspended in 2 liters of 2xTY containing100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phageare prepared as described in International Publication No. WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene m particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneRI protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2xTY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 10¹³ transducing units/rnl(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCI, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG 1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., International Publication No. WO 92/01047) and then by sequencing.These ELISA positive clones may also be further characterized bytechniques known in the art, such as, for example, epitope mapping,binding affinity, receptor signal transduction, ability to block orcompetitively inhibit antibody/antigen binding, and competitiveagonistic or antagonistic activity.

Example 11 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting withdiabetes mellitus is isolated. cDNA is then generated from these RNAsamples using protocols known in the art. (See, Sambrook.) The cDNA isthen used as a template for PCR, employing primers surrounding regionsof interest in SEQ ID NO:X; and/or the nucleotide sequence of the cDNAcontained in ATCC Deposit No:Z. Suggested PCR conditions consist of 35cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degreesC.; and 60-120 seconds at 70 degrees C., using buffer solutionsdescribed in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase(Epicentre Technologies). The intron-exon boundaries of selected exonsis also determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations are then cloned andsequenced to validate the results of the direct sequencing.

PCR products are cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991)). Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 12 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 μg/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described in Example 10. The wells are blocked sothat non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbound polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbound conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 13 Formulation

The invention also provides methods of preventing, treating and/orameliorating diabetes mellitus by administration to a subject of aneffective amount of a Therapeutic. By therapeutic is meantpolynucleotides or polypeptides of the invention (including fragmentsand variants), agonists or antagonists thereof, and/or antibodiesthereto, in combination with a pharmaceutically acceptable carrier type(e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about lug/kg/day to 10 mg/kg/day of patient body weight, although, asnoted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials. (such as, forexample, semi-permeable polymer matrices in the form of shaped articles,e.g., films, or mirocapsules), suitable hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, andsparingly soluble derivatives (such as, for example, a sparingly solublesalt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2- hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, polypeptide, polynucleotide, and antibodycompositions of the invention are formulated in a biodegradable,polymeric drug delivery system, for example as described in U.S. Pat.Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and5,487,897 and in International Publication Numbers WO01/35929,WO00/24374, and WO00/06117 which are hereby incorporated by reference intheir entirety. In specific preferred embodiments the polypeptide,polynucleotide, and antibody compositions of the invention areformulated using the ATRIGEL® Biodegradable System of AtrixLaboratories, Inc. (Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulationof polypeptide, polynucleotide, and antibody compositions, include butare not limited to, polylactides, polyglycolides, polycaprolactones,polyanhydrides, polyamides, polyurethanes, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(amino acids),poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone,polyethylene glycol, polyhydroxycellulose, chitin, chitosan, andcopolymers, terpolymers, or combinations or mixtures of the abovematerials. The preferred polymers are those that have a lower degree ofcrystallization and are more hydrophobic. These polymers and copolymersare more soluble in the biocompatible solvents than the highlycrystalline polymers such as polyglycolide and chitin which also have ahigh degree of hydrogen-bonding. Preferred materials with the desiredsolubility parameters are the polylactides, polycaprolactones, andcopolymers of these with glycolide in which there are more amorphousregions to enhance solubility. In specific preferred embodiments, thebiodegradable polymers which can be used in the formulation ofpolypeptide, polynucleotide, and antibody compositions arepoly(lactide-co-glycolides). Polymer properties such as molecularweight, hydrophobicity, and lactide/glycolide ratio may be modified toobtain the desired polypeptide, polynucleotide, or antibody releaseprofile (See, e.g., Ravivarapu et al., Journal of PharmaceuticalSciences 89:732-741 (2000), which is hereby incorporated by reference inits entirety).

It is also preferred that the solvent for the biodegradable polymer benon-toxic, water miscible, and otherwise biocompatible. Examples of suchsolvents include, but are not limited to, N-methyl-2-pyrrolidone,2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, andtetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone;C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate;alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such asdimethylformamide, dimethylacetamide and caprolactam; C3 to C20 etherssuch as tetrahydrofuran, or solketal; tweens, triacetin, propylenecarbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,1-dodecylazacycloheptan-2-one, Other preferred solvents are benzylalchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate,glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleicacid, polyethylene glycol, propylene carbonate, and triethyl citrate.The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone,dimethyl sulfoxide, triacetin, and propylene carbonate because of thesolvating ability and their compatibility.

Additionally, formulations comprising polypeptide, polynucleotide, andantibody compositions and a biodegradable polymer may also includerelease-rate modification agents and/or pore-forming agents. Examples ofrelease-rate modification agents include, but are not limited to, fattyacids, triglycerides, other like hydrophobic compounds, organicsolvents, plasticizing compounds and hydrophilic compounds. Suitablerelease rate modification agents include, for example, esters of mono-,di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methylacetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutylphthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate,dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyltriethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and thelike; polyhydroxy alcohols, such as propylene glycol, polyethyleneglycol, glycerin, sorbitol, and the like; fatty acids; triesters ofglycerol, such as triglycerides, epoxidized soybean oil, and otherepoxidized vegetable oils; sterols, such as cholesterol; alcohols, suchas C.sub.6-C.sub.12 alkanols, 2-ethoxyethanol. The release ratemodification agent may be used singly or in combination with other suchagents. Suitable combinations of release rate modification agentsinclude, but are not limited to, glycerin/propylene glycol,sorbitolglycerine, ethylene oxidelpropylene oxide, butyleneglycolladipic acid, and the like. Preferred release rate modificationagents include, but are not limited to, dimethyl citrate, triethylcitrate, ethyl heptanoate, glycerin, and hexanediol. Suitablepore-forming agents that may be used in the polymer composition include,but are not limited to, sugars such as sucrose and dextrose, salts suchas sodium chloride and sodium carbonate, polymers such ashydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol,and polyvinylpyrrolidone. Solid crystals that will provide a definedpore size, such as salt or sugar, are preferred.

In specific preferred embodiments the polypeptide, polynucleotide, andantibody compositions of the invention are formulated using the BEMA™BioErodible Mucoadhesive System, MCA™ MucoCutaneous Absorption System,SMP™ Solvent MicroParticle System, or BCP™ BioCompatible Polymer Systemof Atrix Laboratories, Inc. (Fort Collins, Colo.).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 mi ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG (e.g., TBERACYS®), MPL and nonviable prepartionsof Corynebacterium parvum. In a specific embodiment, Therapeutics of theinvention are administered in combination with alum. In another specificembodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, μluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, chemotherapeutic agents, antibiotics,steroidal and non-steroidal anti-inflammatories, conventionalimmunotherapeutic agents, and/or therapeutic treatments described below.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with an anticoagulant. Anticoagulants that may beadministered with the compositions of the invention include, but are notlimited to, heparin, low molecular weight heparin, warfarin sodium(e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g.,MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™),indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate(e.g., TROMEXAN™), and aspirin. In a specific embodiment, compositionsof the invention are administered in combination with heparin and/orwarfarin. In another specific embodiment, compositions of the inventionare administered in combination with warfarin. In another specificembodiment, compositions of the invention are administered incombination with warfarin and aspirin. In another specific embodiment,compositions of the invention are administered in combination withheparin. In another specific embodiment, compositions of the inventionare administered in combination with heparin and aspirin.

In another embodiment, the Therapeutics of the invention areadministered in combination with thrombolytic drugs. Thrombolytic drugsthat may be administered with the compositions of the invention include,but are not limited to, plasminogen, lys-plasminogen,alpha2-antiplasmin, streptokinae (e.g., KABIKMASE™), antiresplace (e.g.,EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™),urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chainurokinase), and aminocaproic acid (e.g., AMICAR™). In a specificembodiment, compositions of the invention are administered incombination with tissue plasminogen activator and aspirin.

In another embodiment, the Therapeutics of the invention areadministered in combination with antiplatelet drugs. Antiplatelet drugsthat may be administered with the compositions of the invention include,but are not limited to, aspirin, dipyridamole (e.g., PERSANTINE™), andticlopidine (e.g., TICLID™).

In specific embodiments, the use of anti-coagulants, thrombolytic and/orantiplatelet drugs in combination with Therapeutics of the invention iscontemplated for the detection, prevention, diagnosis, prognostication,treatment, and/or amelioration of thrombosis, arterial thrombosis,venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis,myocardial infarction, transient ischemic attack, unstable angina. Inspecific embodiments, the use of anticoagulants, thrombolytic drugsand/or antiplatelet drugs in combination with Therapeutics of theinvention is contemplated for the prevention of occulsion of saphenousgrafts, for reducing the risk of periprocedural thrombosis as mightaccompany angioplasty procedures, for reducing the risk of stroke inpatients with atrial fibrillation including nonrheumatic atrialfibrillation, for reducing the risk of embolism associated withmechanical heart valves and or mitral valves disease. Other uses for thetherapeutics of the invention, alone or in combination withantiplatelet, anticoagulant, and/or thrombolytic drugs, include, but arenot limited to, the prevention of occlusions in extracorporeal devices(e.g., intravascular canulas, vascular access shunts in hemodialysispatients, hemodialysis machines, and cardiopulmonary bypass machines).

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may beadministered in combination with the Therapeutics of the invention,include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™(didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T),EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™

iraine), and SUSTIVA™ (eravirenz). Protease inhibitors that may beadministered in

nation with the Therapeutics of the invention, include, but are notlimited to, CRIXIVAN™

avir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™(nelfinavir). In

ific embodiment, antiretroviral agents, nucleoside reverse transcriptaseinhibitors, non-

side reverse transcriptase inhibitors, and/or protease inhibitors may beused in any

nation with Therapeutics of the invention to treat AIDS and/or toprevent or treat HIV

ion.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI;

sle/Abbott; COVIRACIL™ (emtricitabine/FFC; structurally related tolamivudine (3TC) but

- to 10-fold greater activity in vitro; Triangle/Abbott); dOTC(BCH-10652, also structurally

to larnivudine but retains activity against a substantial proportion oflamivudine-resistant

s; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy byFDA; Gilead

es); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; itsactive form is

-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active

olite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activityagainst AZT/3TC-

nt virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159U89; GlaxoWellcome

CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); andS-acyl-2-thioethyl (SATE)-

g prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of

EPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a nextgeneration

I with activity against viruses containing the K103N mutation; Agouron);PNU-142721 (has

50-fold greater activity than its predecessor delavirdine and is activeagainst K103N

its; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generationderivatives of

enz, designed to be active against viruses with the K103N mutation;DuPont); GW420867X

25-fold greater activity than HBY097 and is active against K103Nmutants; Glaxo

ome); CALANOLIDE A (naturally occurring agent from the latex tree;active against

s containing either or both the Y181C and K103N mutations); and Propolis(WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott

atories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™(PNU-

0, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (anonpeptidic

ropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-MyersSquibb); L-756,423 (an

vir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont);AG-1776 (a

lornimetic with in vitro activity against protease inhibitor-resistantviruses; Agouron); VX-

W-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome);CGP61755

); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX404C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonistssuch as RANTES (968), AOP-RANTES, NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetoragonists such as RANTES, SDF-1, MIP-1, MIP-1,, etc., may also inhibitfusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compunds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha asVX497 (Vertex); and mycopholic acids such as CellCept (mycophenolatemofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120,gp120/soluble CD4 complex, Delta JR-FL protein, branched syntheticpeptide derived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(genetically modified CC chemokines targetted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as theanti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9,PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4,the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d,447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-αantibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptoragonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl,3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); andantioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO99/56764).

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIIMTHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, the Therapeutics of the invention are administeredin combination with immunestimulants. Immunostimulants that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, levamisole (e.g., ERGAMISOL™),isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), andinterleukins (e.g., IL-2).

In other embodiments, Therapeutics of the invention are administered incombination with immunosuppressive agents. Immunosuppressive agents thatmay be administered in combination with the Therapeutics of theinvention include, but are not limited to, steroids, cyclosporine,cyclosporine analogs, cyclophosphamide methylprednisone, prednisone,azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressiveagents that act by suppressing the function of responding T cells. Otherimmunosuppressive agents that may be administered in combination withthe Therapeutics of the invention include, but are not limited to,prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin,leflunomide, mizoribine (BREDINI), brequinar, deoxyspergualin, andazaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™,NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus),CELLCEPT® (mycophenolate motefil, of which the active metabolite ismycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids,adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™(prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™(methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™(antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment,Therapeutics of the invention are administered in combination withintravenous immune globulin preparations in transplantation therapy(e.g., bone marrow transplant).

In certain embodiments, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, corticosteroids (e.g.betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, and triamcinolone),nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal,etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam,nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, (1991)); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, dL-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChlMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,(1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-316, (1992)); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J. Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J. Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administed in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositons of the invention include, but are not limited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositons of the inventioninclude, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not limited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/ Pharmacia Upjohn, Bridgewater,N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act toindirectly inhibit angiogenesis. Examples of indirect inhibitors ofangiogenesis which may be administered in combination with thecompositons of the invention include, but are not limited to, IM-862(Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley,N.J.), and Pentosan polysulfate (Georgetown University, Washington,D.C.).

In particular embodiments, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

In another embodiment, the polynucleotides encoding a polypeptide of thepresent invention are administered in combination with an angiogenicprotein, or polynucleotides encoding an angiogenic protein. Examples ofangiogenic proteins that may be administered with the compositions ofthe invention include, but are not limited to, acidic and basicfibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growthfactor alpha and beta, platelet-derived endothelial cell growth factor,platelet-derived growth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin-like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

In additional embodiments, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to alkylating agents suchas nitrogen mustards (for example, Mechlorethamine, cyclophosphamide,Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), andChlorambucil), ethylenimines and methylmelamines (for example,Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example,Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine(CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)),triazenes (for example, Dacarbazine (DTIC;dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example,Methotrexate (amethopterin)), pyrimidine analogs (for example,Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine;FudR), and Cytarabine (cytosine arabinoside)), purine analogs andrelated inhibitors (for example, Mercaptopurine (6-mercaptopurine;6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin(2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB,vinblastine sulfate)) and Vincristine (vincristine sulfate)),epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics(for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin;rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), andMitomycin (mitomycin C), enzymes (for example, L-Asparaginase),biological response modifiers (for example, Interferon-alpha andinterferon-alpha-2b), platinum coordination compounds (for example,Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone),substituted ureas (for example, Hydroxyurea), methylhydrazinederivatives (for example, Procarbazine (N-methylhydrazine; MIH),adrenocorticosteroids (for example, Prednisone), progestins (forexample, Hydroxyprogesterone caproate, Medroxyprogesterone,Medroxyprogesterone acetate, and Megestrol acetate), estrogens (forexample, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate,Estradiol, and Ethinyl estradiol), antiestrogens (for example,Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone),antiandrogens (for example, Flutamide), gonadotropin-releasing horomoneanalogs (for example, Leuprolide), other hormones and hormone analogs(for example, methyltestosterone, estramustine, estramustine phosphatesodium, chlorotrianisene, and testolactone), and others (for example,dicarbazine, glutamic acid, and mitotane).

In one embodiment, the compositions of the invention are administered incombination with one or more of the following drugs: infliximab (alsoknown as Remicader™ Centocor, Inc.), Trocade (Roche, RO-32-3555),Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™(an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.) Ina specific embodiment, compositions of the invention are administered incombination with CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone) or combination of one or more of the components of CHOP. Inone embodiment, the compositions of the invention are administered incombination with anti-CD20 antibodies, human monoclonal anti-CD20antibodies. In another embodiment, the compositions of the invention areadministered in combination with anti-CD20 antibodies and CHOP, oranti-CD20 antibodies and any combination of one or more of thecomponents of CHOP, particularly cyclophospharnide and/or prednisone. Ina specific embodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. In a specific embodiment,compositions of the invention are administered in combination withtositumomab. In a further embodiment, compositions of the invention areadministered with tositumomab and CHOP, or tositumomab and anycombination of one or more of the components of CHOP, particularlycyclophospharnide and/or prednisone. The anti-CD20 antibodies mayoptionally be associated with radioisotopes, toxins or cytotoxicprodrugs.

In another specific embodiment, the compositions of the invention areadministered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radisotopes. Particularly preferred isotopesare ⁹⁰Y and ¹¹¹In.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, lL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNP, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TRANK, TR9 (International Publication No. WO 98/56892),TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PIGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in Gernan Patent Number DE19639601. The above mentionedreferences are herein incorporated by reference in their entireties.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-I 1, FGF-12, FGF-13,FGF-14, and FGF-15.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to,granulocyte macrophage colony stimulating factor (GM-CSF)(sargramostirn, LEUKINE™, PROKINE™), granulocyte colony stimulatingfactor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulatingfactor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™),stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colonystimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins,especially any one or more of IL-1 through IL-12, interferon-gamma, orthrombopoietin.

In certain embodiments, Therapeutics of the present invention areadministered in combination with adrenergic blockers, such as, forexample, acebutolol, atenolol, betaxolol, bisoprolol, carteolol,labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol,propranolol, sotalol, and timolol.

In another embodiment, the Therapeutics of the invention areadministered in combination with an antiarrhythmic drug (e.g.,adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin,diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine,moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone,propranolol, quinidine, sotalol, tocainide, and verapamil).

In another embodiment, the Therapeutics of the invention areadministered in combination with diuretic agents, such as carbonicanhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenarnide, andmethazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol,and urea), diuretics that inhibit Na⁺-K⁺-2Cl⁻ symport (e.g., furosemide,bumetanide, azosemide, piretanide, tripamide, ethacrynic acid,muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g.,bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide,chlorthalidone, indapamide, metolazone, and quinethazone), potassiumsparing diuretics (e.g., amiloride and triamterene), andmineralcorticoid receptor antagonists (e.g., spironolactone, canrenone,and potassium canrenoate).

In one embodiment, the Therapeutics of the invention are administered incombination with treatments for endocrine and/or hormone imbalancedisorders. Treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, ¹²⁷I, radioactive isotopes of iodinesuch as ¹³¹I and ¹²³I; recombinant growth hormone, such as HUMATROPE™(recombinant somatropin); growth hormone analogs such as PROTROPIN™(somatrem); dopamine agonists such as PARLODEL™ (bromocriptine);somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropinpreparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionicgonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™(urofollitropin (uFSH)); synthetic human gonadotropin releasing hormonepreparations such as FACTREL™ and LUTREPULSE™ (gonadorelinhydrochloride); synthetic gonadotropin agonists such as LUPRON™(leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™(nafarelin acetate), and ZOLADEX™ (goserelin acetate); syntheticpreparations of thyrotropin-releasing hormone such as RELEFACT TRH™ andTHYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™;synthetic preparations of the sodium salts of the natural isomers ofthyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™(levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroinesodium), and THYROLAR™ (liotrix); antithyroid compounds such as6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazoleand TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole);beta-adrenergic receptor antagonists such as propranolol and esmolol;Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrastagents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodiumipodate).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, estrogens or congugated estrogens suchas ESTRACE™ (estradiol), ESTNYL™ (ethinyl estradiol), PREMARIN™,ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™(quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™(estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™(estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen),SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™(hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™(medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™(megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ andAYGESTIN™ (norethindrone acetate); progesterone implants such asNORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins suchas RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™(norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device thatreleases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™,NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinylestradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ andTREPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™(ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodioldiacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™(norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinylestradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinylestradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), andOVRETTE™ (norgestrel).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, testosterone esters such as methenoloneacetate and testosterone undecanoate; parenteral and oral androgens suchas TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate),DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosteronecypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETONMETHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™(oxandrolone); testosterone transdermal systems such as TESTODERM™;androgen receptor antagonist and 5-alpha-reductase inhibitors such asANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™(finasteride); adrenocorticotropic hormone preparations such asCORTROSYN™ (cosyntropin); adrenocortical steroids and their syntheticanalogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™(amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate),CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasonebenzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™(betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasonesodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasonevalerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolonepivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)),HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™(cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol(hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™(cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol(hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate),DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone),DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate),DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodiumphosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEFACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™(flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™(fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™(flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone),MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™(methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™(methylprednisolone sodium succinate), ELOCON™ (mometasone furoate),HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone),ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodiumphosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™(prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™(triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™(triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide);inhibitors of biosynthesis and action of adrenocortical steroids such asCYTADREN™ (aminoglutethirnide), NIZORAL™ (ketoconazole), MODRASTANE™(trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or humaninsulin or mixtures thereof; insulin analogs; recombinant human insulinsuch as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ andTOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide,MICRONASE™, DIDBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide),and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone,pioglitazone, and alpha-glucosidase inhibitors; bovine or porcineglucagon; somatostatins such as SANDOSTATIN™ (octreotide); anddiazoxides such as PROGLYCEM™ (diazoxide).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with drugs effective in treating irondeficiency and hypochromic anemias, including but not limited to,ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g.,FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-ironcomplex (e.g., NEFEREX™), iron dextran injection (e.g., INFED™), cupricsulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection(e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g.,FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor)or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.

In another embodiment, Therapeutics of the invention are administered incombination with vasodilating agents and/or calcium channel blockingagents. Vasodilating agents that may be administered with theTherapeutics of the invention include, but are not limited to,Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine,isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat,fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril,spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbidedinitrate, isosorbide mononitrate, and nitroglycerin). Examples ofcalcium channel blocking agents that may be administered in combinationwith the Therapeutics of the invention include, but are not limited toamlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine,nicardipine, nifedipine, nimodipine, and verapamil.

In certain embodiments, the Therapeutics of the invention areadministered in combination with treatments for gastrointestinaldisorders. Treatments for gastrointestinal disorders that may beadministered with the Therapeutic of the invention include, but are notlimited to, H₂ histamine receptor antagonists (e.g., TAGAMET™(cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™(nizatidine)); inhibitors of H⁺, K⁺ATPase (e.g., PREVACID™(lansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g.,PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate));various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™(misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g.,surfactant laxatives, stimulant laxatives, saline and osmoticlaxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate),MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)),synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide),antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetronhydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine,perphenazine, prochlorperazine, promethazine, thiethylperazine,triflupromazine, domperidone, haloperidol, droperidol,trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, andnabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide andchlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholicacid; and pancreatic enzyme preparations such as pancreatin andpancrelipase.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 14 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of polypeptides (including agoniststhereto), and/or antibodies of the invention. Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a polypeptide of the present invention in anindividual may be treated by administering agonists of said polypeptide.Thus, the invention also provides a method of treatment of an individualin need of an increased level of the polypeptide comprisingadministering to such an individual a Therapeutic comprising an amountof the agonist (including polypeptides and antibodies of the presentinvention) to increase the activity level of the polypeptide in such anindividual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the agonist for six consecutive days. Theexact details of the dosing scheme, based on administration andformulation, are provided in Example 13.

Example 15 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, due to a variety ofetiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels of apolypeptide is administered intravenously antisense polynucleotides at0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment isrepeated after a 7-day rest period if the treatment was well tolerated.The antisense polynucleotides of the present invention can be formulatedusing techniques and formulations described herein (e.g. see Example13), or otherwise known in the art.

Example 16 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asubconfluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 17 Gene Therapy Using Endogenous Genes Corresponding ToPolynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel, then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′ end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindIII site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 18 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to prevent, treat, and/or ameliorate diabetes mellitus. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) sequences into an animal to increase ordecrease the expression of the polypeptide. The polynucleotide of thepresent invention may be operatively linked to (i.e., associated with) apromoter or any other genetic elements necessary for the expression ofthe polypeptide by the target tissue. Such gene therapy and deliverytechniques and methods are known in the art, see, for example,WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, 5,705,151, 5,580,859;Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al.,Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord.7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996);Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated hereinby reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapy techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within an animal, including muscle, skin, brain, lung, liver,spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage,pancreas, kidney, gall bladder, stomach, intestine, testis, ovary,uterus, rectum, nervous system, eye, gland, and connective tissue.Interstitial space of the tissues comprises the intercellular fluid,mucopolysaccharide matrix among the reticular fibers of organ tissues,elastic fibers in the walls of vessels or chambers, collagen fibers offibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be used toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 19 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying conditionsand/or disorders associated with aberrant expression, and in screeningfor compounds effective in ameliorating such conditions and/ordisorders.

Example 20 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (e.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying conditions and/or disorders associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

Example 21 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

Diabetic db+/db+Mouse Model.

To demonstrate that an agonist or antagonist of the inventionaccelerates the healing process, the genetically diabetic mouse model ofwound healing is used. The full thickness wound healing model in thedb+/db+mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al., J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have-a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 16 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BLKsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

An agonist or antagonist of the invention is administered using at arange different doses, from 4 mg to 500 mg per wound per day for 8 daysin vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with an agonist or antagonist of the invention. Thisassessment included verification of the presence of cell accumulation,inflanmmatory cells, capillaries, fibroblasts, re-epithelialization andepidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235(1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975);Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

To demonstrate that an agonist or antagonist of the invention canaccelerate the healing process, the effects of multiple topicalapplications of the agonist or antagonist on full thickness excisionalskin wounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The agonist or antagonist of the invention is administered using at arange different doses, from 4 mg to 500 mg per wound per day for 8 daysin vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Three groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith an agonist or antagonist of the invention. A calibrated lensmicrometer is used by a blinded observer to determine the distance ofthe wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity of agonists orantagonists of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the activity ofpolynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 22 Production Of Polypeptide of the Invention ForHigh-Throughput Screening Assays

The following protocol produces a supernatant containing polypeptide ofthe present invention to be tested. This supernatant can then be used inthe Screening Assays described in Examples 24-27.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503FBiowhittaker)/1× Penstrep(17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multihannel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples8-10, into an appropriately labeled 96-well round bottom plate. With amultihannel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multihannel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37 degree C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2 (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄-H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid ; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Olcic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamineand 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degree C. for 45 or 72 hours depending on the mediaused: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 24-27.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the polypeptide of the present invention directly (e.g., asa secreted protein) or by polypeptide of the present invention inducingexpression of other proteins, which are then secreted into thesupernatant. Thus, the invention further provides a method ofidentifying the protein in the supernatant characterized by an activityin a particular assay.

Example 23 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995)). A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xaa-Trp-Ser (SEQ ID NO: 2)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway. Therefore, activation of the Jaks-STATs pathway, reflected bythe binding of the GAS or the ISRE element, can be used to indicateproteins involved in the proliferation and differentiation of cells. Forexample, growth factors and cytokines are known to activate theJaks-STATs pathway (See Table below). Thus, by using GAS elements linkedto reporter molecules, activators of the Jaks-STATs pathway can beidentified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISREIFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11 (Pleiotropic) ? + ? ? 1, 3 OnM (Pleiotropic)? + + ? 1, 3 LIF (Pleiotropic) ? + + ? 1, 3 CNTF (Pleiotropic) −/+ + + ?1, 3 G-CSF (Pleiotropic) ? + ? ? 1, 3 IL-12 (Pleiotropic) + − + + 1, 3g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid)− + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GASIL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRFI promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris: (SEQ ID NO: 3)5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′:GCGGCAAGCTTGCAAAGCCTAGGC:3′ (SEQ ID NO:4) PCR amplification is performed using the SV40 promoter templatepresent in the B-gal:promoter plasmid obtained from Clontech. Theresulting PCR fragment is digested with XhoI/Hind III and subcloned intoBLSK2-. (Stratagene.) Sequencing with forward and reverse primersconfirms that the insert contains the following sequence: (SEQ ID NO: 5)5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence, for example, with EGR and NF-KBpromoter sequences. However, many other promoters can be substitutedusing the protocols described in these Examples. For instance, SRE,IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or incombination (e.g., GAS/NF-KB/EGR, GAS/NF-KB3, Il-2/NFAT, or NF-KB/GAS).Similarly, other cell lines can be used to test reporter constructactivity, such as BELA (epithelial), HUVEC (endothelial), Reh (Bcell),Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 24 Assay for SEAP Acdivity

SEAP activity is assayed using the Tropix Phospholight Kit (Cat. BP400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see the Table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on a luminometer, thus one should treat 5 plates ateach time and start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity. Reaction Buffer Formulation: # of plates Rxn buffer diluent(ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 854.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.529 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 1859.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 21510.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 24512.25 48 250 12.5 49 255 12.75 50 260 13

Example 25 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 degreesC. water bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley Cell Wash with 200 ul, followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4 . The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by the a molecule, either polypeptide of the present invention ora molecule induced by polypeptide of the present invention, which hasresulted in an increase in the intracellular Ca⁺⁺ concentration.

Example 26 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, Ick, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether polypeptide of the presentinvention or a molecule induced by polypeptide of the present inventionis capable of activating tyrosine kinase signal transduction pathways isof interest. Therefore, the following protocol is designed to identifysuch molecules capable of activating the tyrosine kinase signaltransduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford,Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford,Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 22, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of proteaseinhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.)) is added to each well and the plate is shaken on a rotatingshaker for 5 minutes at 4° C. The plate is then placed in a vacuumtransfer manifold and the extract filtered through the 0.45 mm membranebottoms of each well using house vacuum. Extracts are collected in a96-well catch/assay plate in the bottom of the vacuum manifold andimmediately placed on ice. To obtain extracts clarified bycentrifugation, the content of each well, after detergent solubilizationfor 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C.at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degree C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degree C. for 20 min. This allows the streptavidin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml))to each well and incubate at 37 degree C. for one hour. Wash the well asabove.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 27 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or complement to the assay of proteintyrosine kinase activity described in Example 26, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 22 for 5-20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Waflac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by polypeptide of the presentinvention or a molecule induced by polypeptide of the present invention.

Example 28 Detection of Inhibition of a Mixed Lymphocyte Reaction

This assay can be used to detect and evaluate inhibition of a MixedLymphocyte Reaction (MLR) by gene products (e.g., isolatedpolypeptides). Inhibition of a MLR may be due to a direct effect on cellproliferation and viability, modulation of costimulatory molecules oninteracting cells, modulation of adhesiveness between lymphocytes andaccessory cells, or modulation of cytokine production by accessorycells. Multiple cells may be targeted by these polypeptides since theperipheral blood mononuclear fraction used in this assay includes T, Band natural killer lymphocytes, as well as monocytes and dendriticcells.

Polypeptides of interest found to inhibit the MLR may find applicationin diseases associated with lymphocyte and monocyte activation orproliferation. These include, but are not limited to, diseases such asasthma, arthritis, diabetes, inflammatory skin conditions, psoriasis,eczema, systemic lupus erythematosus, multiple sclerosis,glomerulonephritis, inflammatory bowel disease, crohn's disease,ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease,host vs. graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors are purified by density gradientcentrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from twodonors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies,Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCsfrom a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters ofPBMCs from each donor is added to wells of a 96-well round bottommicrotiter plate. Dilutions of test materials (50 μl) is added intriplicate to microtiter wells. Test samples (of the protein ofinterest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems,Minneapolis, Minn., catalog number 202-IL) is added to a finalconcentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11,catalog number MAB379) is added to a final concentration of 10 μg/ml.Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H]thymidine is added to wells for the last 16 hrs of culture. Cells areharvested and thymidine incorporation determined using a PackardTopCount. Data is expressed as the mean and standard deviation oftriplicate determinations.

Samples of the protein of interest are screened in separate experimentsand compared to the negative control treatment, anti-CD4 mAb, whichinhibits proliferation of lymphocytes and the positive controltreatment, IL-2 (either as recombinant material or supernatant), whichenhances proliferation of lymphocytes.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof.

Example 29 Assays for Protease Activity

The following assay may be used to assess protease activity of thepolypeptides of the invention.

Gelatin and casein zymography are performed essentially as described(Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al.,Journal of Urology, 149:653-658 (1993)). Samples are run on 10%polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3at 37° C. 5 to 16 hours. After staining in amido black areas ofproteolysis apear as clear areas agains the blue-black background.Trypsin (Sigma T8642) is used as a positive control.

Protease activity is also determined by monitoring the cleavage ofn-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500. Reactions areset up in (25 mMNaPO₄,1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples areadded and the change in adsorbance at 260nm is monitored on the BeckmanDU-6 spectrophotometer in the time-drive mode. Trypsin is used as apositive control.

Additional assays based upon the release of acid-soluble peptides fromcasein or hemoglobin measured as adsorbance at 280 nm orcolorimetrically using the Folin method are performed as described inBergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assaysinvolve the solubilization of chromogenic substrates (Ward, AppliedScience, 251-317 (1983)).

Example 30 Identifying Serine Protease Substrate Specificity

Methods known in the art or described herein may be used to determinethe substrate specificity of the polypeptides of the present inventionhaving serine protease activity. A preferred method of determiningsubstrate specificity is by the use of positional scanning syntheticcombinatorial libraries as described in GB2 324 529 (incorporated hereinin its entirety).

Example 31 Ligand Binding Assays

The following assay may be used to assess ligand binding activity of thepolypeptides of the invention.

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. The purifiedligand for a polypeptide is radiolabeled to high specific activity(50-2000 Ci/mmol) for binding studies. A determination is then made thatthe process of radiolabeling does not diminish the activity of theligand towards its polypeptide. Assay conditions for buffers, ions, pHand other modulators such as nucleotides are optimized to establish aworkable signal to noise ratio for both membrane and whole cellpolypeptide sources. For these assays, specific polypeptide binding isdefined as total associated radioactivity minus the radioactivitymeasured in the presence of an excess of unlabeled competing ligand.Where possible, more than one competing ligand is used to defineresidual nonspecific binding.

Example 32 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding thepolypeptides of the invention are synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus ooctyesin response polypeptides and polypeptide agonist exposure. Recordingsare made in Ca2+ free Barth's medium at room temperature. The Xenopussystem can be used to screen known ligands and tissue/cell extracts foractivating ligands.

Example 33 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results inextrusion of small amounts of acid from a cell. The acid formed islargely as a result of the increased metabolic activity required to fuelthe intracellular signaling process. The pH changes in the mediasurrounding the cell are very small but are detectable by the CYTOSENSORmicrophysiometer (Molecular Devices Ltd., Menlo Park, Calif.). TheCYTOSENSOR is thus capable of detecting the activation of polypeptidewhich is coupled to an energy utilizing intracellular signaling pathway.

Example 34 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, asyet, no cognate activating ligand (agonist). Thus, active ligands forthese receptors may not be included within the ligands banks asidentified to date. Accordingly, the polypeptides of the invention canalso be functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify its natural ligands. Extracts that produce positivefunctional responses can be sequentially subfractionated until anactivating ligand is isolated and identified.

Example 35 Calcium and cAMP Functional Assays

Seven transmembrane receptors which are expressed in HEK 293 cells havebeen shown to be coupled functionally to activation of PLC and calciummobilization and/or cAMP stimulation or inhibition. Basal calcium levelsin the HEK 293 cells in receptor-transfected or vector control cellswere observed to be in the normal, 100 nM to 200 nM, range. HEK 293cells expressing recombinant receptors are loaded with fura 2 and in asingle day >150 selected ligands or tissue/cell extracts are evaluatedfor agonist induced calcium mobilization. Similarly, HEK 293 cellsexpressing recombinant receptors are evaluated for the stimulation orinhibition of cAMP production using standard cAMP quantitation assays.Agonists presenting a calcium transient or cAMP fluctuation are testedin vector control cells to determine if the response is unique to thetransfected cells expressing receptor.

Example 36 ATP-binding Assay

The following assay may be used to assess ATP-binding activity ofpolypeptides of the invention.

ATP-binding activity of the polypeptides of the invention may bedetected using the ATP-binding assay described in U.S. Pat. No.5,858,719, which is herein incorporated by reference in its entirety.Briefly, ATP-binding to polypeptides of the invention is measured viaphotoaffinity labeling with 8-azido-ATP in a competition assay. Reactionmixtures containing 1 mg/ml of the ABC transport protein of the presentinvention are incubated with varying concentrations of ATP, or thenon-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at4° C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus8-azido-ATP (³²P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to afinal concentration of 100 μM and 0.5 ml aliquots are placed in thewells of a porcelain spot plate on ice. The plate is irradiated using ashort wave 254 nm UV lamp at a distance of 2.5 cm from the plate for twoone-minute intervals with a one-minute cooling interval in between. Thereaction is stopped by addition of dithiothreitol to a finalconcentration of 2 mM. The incubations are subjected to SDS-PAGEelectrophoresis, dried, and autoradiographed. Protein bandscorresponding to the particular polypeptides of the invention areexcised, and the radioactivity quantified. A decrease in radioactivitywith increasing ATP or adenly-5′-imidodiphosphate provides a measure ofATP affinity to the polypeptides.

Example 37 Small Molecule Screening

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and polypeptide of the invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe invention. These methods comprise contacting such an agent with apolypeptide of the invention or fragment thereof and assaying for thepresence of a complex between the agent and the polypeptide or fragmentthereof, by methods well known in the art. In such a competitive bindingassay, the agents to screen are typically labeled. Following incubation,free agent is separated from that present in bound form, and the amountof free or uncomplexed label is a measure of the ability of a particularagent to bind to the polypeptides of the invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe invention, and is described in great detail in European PatentApplication 84/03564, published on Sep. 13, 1984, which is hereinincorporated by reference in its entirety. Briefly stated, large numbersof different small molecule test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The testcompounds are reacted with polypeptides of the invention and washed.Bound polypeptides are then detected by methods well known in the art.Purified polypeptides are coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies may be used to capture the peptide and inmmobilize it on thesolid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the invention specifically compete with a test compound for bindingto the polypeptides or fragments thereof. In this manner, the antibodiesare used to detect the presence of any peptide which shares one or moreantigenic epitopes with a polypeptide of the invention.

Example 38 Phosphorylation Assay

In order to assay for phosphorylation activity of the polypeptides ofthe invention, a phosphorylation assay as described in U.S. Pat. No.5,958,405 (which is herein incorporated by reference) is utilized.Briefly, phosphorylation activity may be measured by phosphorylation ofa protein substrate using gamma-labeled ³²P-ATP and quantitation of theincorporated radioactivity using a gamma radioisotope counter. Thepolypeptides of the invention are incubated with the protein substrate,³²P-ATP, and a kinase buffer. The ³²P incorporated into the substrate isthen separated from free ³²P-ATP by electrophoresis, and theincorporated ³²P is counted and compared to a negative control.Radioactivity counts above the negative control are indicative ofphosphorylation activity of the polypeptides of the invention.

Example 39 Detection of Phosphorylation Activity (Activation) of thePolypeptides of the Invention in the Presence of Polypeptide Ligands

Methods known in the art or described herein may be used to determinethe phosphorylation activity of the polypeptides of the invention. Apreferred method of determining phosphorylation activity is by the useof the tyrosine phosphorylation assay as described in U.S. Pat. No.5,817,471 (incorporated herein by reference).

Example 40 Identification Of Signal Transduction Proteins That InteractWith Polypeptides Of The Present Invention

The purified polypeptides of the invention are research tools for theidentification, characterization and purification of additional signaltransduction pathway proteins or receptor proteins. Briefly, labeledpolypeptides of the invention are useful as reagents for thepurification of molecules with which it interacts. In one embodiment ofaffinity purification, polypeptides of the invention are covalentlycoupled to a chromatography column. Cell-free extract derived fromputative target cells, such as carcinoma tissues, is passed over thecolumn, and molecules with appropriate affinity bind to the polypeptidesof the invention. The protein complex is recovered from the column,dissociated, and the recovered molecule subjected to N-terminal proteinsequencing. This amino acid sequence is then used to identify thecaptured molecule or to design degenerate oligonucleotide probes forcloning the relevant gene from an appropriate cDNA library.

Example 41 Assay for Phosphatase Activity

The following assay may be used to assess serine/threonine phosphatase(PTPase) activity of the polypeptides of the invention.

In order to assay for serine/threonine phosphatase (PIPase) activity,assays can be utilized which are widely known to those skilled in theart. For example, the serine/threonine phosphatase (PSPase) activity ismeasured using a PSPase assay kit from New England Biolabs, Inc. Myelinbasic protein (MyBP), a substrate for PSPase, is phosphorylated onserine and threonine residues with cAMP-dependent Protein Kinase in thepresence of [³²P]ATP. Protein serine/threonine phosphatase activity isthen determined by measuring the release of inorganic phosphate from32P-labeled MyBP.

Example 42 Interaction of Serine/Threonine Phosphatases with otherProteins

The polypeptides of the invention with serine/threonine phosphataseactivity as determined in Example 41 are research tools for theidentification, characterization and purification of additionalinteracting proteins or receptor proteins, or other signal transductionpathway proteins. Briefly, labeled polypeptide(s) of the invention isuseful as a reagent for the purification of molecules with which itinteracts. In one embodiment of affinity purification, polypeptide ofthe invention is covalently coupled to a chromatography column.Cell-free extract derived from putative target cells, such as neural orliver cells, is passed over the column, and molecules with appropriateaffinity bind to the polypeptides of the invention. The polypeptides ofthe invention-complex is recovered from the column, dissociated, and therecovered molecule subjected to N-terminal protein sequencing. Thisamino acid sequence is then used to identify the captured molecule or todesign degenerate oligonucleotide probes for cloning the relevant genefrom an appropriate cDNA library.

Example 43 Assaying for Heparanase Activity

In order to assay for heparanase activity of the polypeptides of theinvention, the heparanase assay described by Vlodavsky et al is utilized(Vlodavsky, I., et al., Nat. Med., 5:793-802 (1999)). Briefly, celllysates, conditioned media or intact cells (1×10⁶ cells per 35-mm dish)are incubated for 18 hrs at 37° C., pH 6.2-6.6, with ³⁵S-labeled ECM orsoluble ECM derived peak I proteoglycans. The incubation medium iscentrifuged and the supernatant is analyzed by gel filtration on aSepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS andtheir radioactivity is measured. Degradation fragments of heparansulfate side chains are eluted from Sepharose 6B at 0.5<K_(av)<0.8 (peakII). Each experiment is done at least three times. Degradation fragmentscorresponding to “peak II,” as described by Vlodavsky et al., isindicative of the activity of the polypeptides of the invention incleaving heparan sulfate.

Example 44 Immobilization of Biomolecules

This example provides a method for the stabilization of polypeptides ofthe invention in non-host cell lipid bilayer constucts (see, e.g., Bieriet al., Nature Biotech 17:1105-1108 (1999), hereby incorporated byreference in its entirety herein) which can be adapted for the study ofpolypeptides of the invention in the various functional assays describedabove. Briefly, carbohydrate-specific chemistry for biotinylation isused to confine a biotin tag to the extracellular domain of thepolypeptides of the invention, thus allowing uniform orientation uponimmobilization. A 50 uM solution of polypeptides of the invention inwashed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACHor 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature(reaction volume, 150 ul). Then the sample is dialyzed (PierceSlidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford Ill.)at 4C first for 5 h, exchanging the buffer after each hour, and finallyfor 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodiumphosphate, pH7). Just before addition into a cuvette, the sample isdiluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mMoctylglucoside).

Example 45 TAQMAN

Quantitative PCR (QPCR). Total RNA from cells in culture are extractedby Trizol separation as recommended by the supplier (LifeTechnologies).(Total RNA is treated with DNase I (Life Technologies) to remove anycontaminating genomic DNA before reverse transcription.) Total RNA (50ng) is used in a one-step, 50 ul, RT-QPCR, consisting of Taqman Buffer A(Perkin-Elmer; 50 mM KCl/10 mM Tris, pH 8.3), 5.5 mM MgCl₂, 240 μM eachdNTP, 0.4 units RNase inhibitor(Promega), 8% glycerol, 0.012% Tween-20,0.05% gelatin, 0.3 uM primers, 0.1 uM probe, 0.025 units Amplitaq Gold(Perkin-Elmer) and 2.5 units Superscript II reverse transcriptase (LifeTechnologies). As a control for genomnic contamination, parallelreactions are setup without reverse transcriptase. The relativeabundance of (unknown) and 18S RNAs are assessed by using the AppliedBiosystems Prism 7700 Sequence Detection System (Livak, K. J., Flood, S.J., Marmaro, J., Giusti, W. & Deetz, K. (1995) PCR Methods Appl. 4,357-362). Reactions are carried out at 48° C. for 30 min, 95° C. for 10min, followed by 40 cycles of 95° C. for 15 s, 60° C. for 1 min.Reactions are performed in triplicate.

Primers (f & r) and FRET probes sets are designed using Primer ExpressSoftware (Perkin-Elmer). Probes are labeled at the 5′-end with thereporter dye 6-FAM and on the 3′-end with the quencher dye TAMRA(Biosource International, Camarillo, Calif. or Perkin-Elmer).

Example 46 Assays for Metalloproteinase Activity

Metalloproteinases (EC 3.4.24.-) are peptide hydrolases which use metalions, such as Zn²⁺, as the catalytic mechanism. Metalloproteinaseactivity of polypeptides of the present invention can be assayedaccording to the following methods.

Proteolysis of alpha-2-macroglobulin

To confirm protease activity, purified polypeptides of the invention aremixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; BoehringerMannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl,10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at 37° C. for1-5 days. Trypsin is used as positive control. Negative controls containonly alpha-2-macroglobulin in assay buffer. The samples are collectedand boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresisthe proteins are visualized by silver staining. Proteolysis is evidentby the appearance of lower molecular weight bands as compared to thenegative control.

Inhibition of alpha-2-macroglobulin proteolysis by inhibitors ofmetalloproteinases Known metalloproteinase inhibitors (metal chelators(EDTA, EGTA, AND HgCl₂), peptide metalloproteinase inhibitors (TIMP-1and TIMP-2), and commercial small molecule MMP inhibitors) are used tocharacterize the proteolytic activity of polypeptides of the invention.The three synthetic MMP inhibitors used are: MMP inhibitor I, [IC₅₀=1.0μM against MMP-1 and MMP-8; IC₅₀=30 μM against MMP-9; IC₅₀=150 μMagainst MMP-3]; MMP-3 (stromelysin-inhibitor I [IC₅₀=5 μM againstMMP-3], and MMP-3 inhibitor II [K_(i)=130 nM against MMP-3]inhibitorsavailable through Calbiochem, catalog #444250, 444218, and 444225,respectively). Briefly, different concentrations of the small moleculeMMP inhibitors are mixed with purified polypeptides of the invention (50μg/ml) in 22.9 μl of 1× HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl,10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at roomtemperature (24° C.) for 2-hr, then 7.1 μl of substratealpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for20-hr. The reactions are stopped by adding 4× sample buffer and boiledimmediately for 5 minutes. After SDS-PAGE, the protein bands arevisualized by silver stain.

Synthetic Fluorogenic Peptide Substrates Cleavage Assay

The substrate specificity for polypeptides of the invention withdemonstrated metalloproteinase activity can be determined usingsynthetic fluorogenic peptide substrates (purchased from BACHEMBioscience Inc). Test substrates include, M-1985, M-2225, M-2105,M-2110, and M-2255. The first four are MMP substrates and the last oneis a substrate of tumor necrosis factor-α (TNF-α) converting enzyme(TACE). All the substrates are prepared in 1:1 dimethyl sulfoxide (DMSO)and water. The stock solutions are 50-500 μM. Fluorescent assays areperformed by using a Perkin Elmer LS 50B luminescence spectrometerequipped with a constant temperature water bath. The excitation λ is 328nm and the emission λ is 393 nnm Briefly, the assay is carried out byincubating 176 μl 1× HEPES buffer (0.2 M NaCl, 10 MM CaCl₂, 0.05%Brij-35 and 50 mM BEPES, pH 7.5) with 4 μl of substrate solution (50 μM)at 25° C. for 15 minutes, and then adding 20 μl of a purifiedpolypeptide of the invention into the assay cuvett. The finalconcentration of substrate is 1 μM. Initial hydrolysis rates aremonitored for 30-min.

Example 47 Characterization of the cDNA contained in a Deposited Plasmid

The size of the cDNA insert contained in a deposited plasmid may beroutinely determined using techniques known in the art, such as PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the cDNA sequence. For example, two primers of 17-30 nucleotidesderived from each end of the cDNA (i.e., hybridizable to the absolute 5′nucleotide or the 3′ nucleotide end of the sequence of SEQ ID NO:X,respectively) are synthesized and used to amplify the cDNA using thedeposited cDNA plasmid as a template. The polymerase chain reaction iscarried out under routine conditions, for instance, in 25 ul of reactionmixture with 0.5 ug of the above cDNA template. A convenient reactionmixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP,dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taqpolymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C.for 1 min) are performed with a Perkin-Elmer Cetus automated thermalcycler. The amplified product is analyzed by agarose gelelectrophoresis. The PCR product is verified to be the selected sequenceby subcloning and sequencing the DNA product.It will be clear that theinvention may be practiced otherwise than as particularly described inthe foregoing description and examples. Numerous modifications andvariations of the present invention are possible in light of the aboveteachings and, therefore, are within the scope of the appended claims.

Incorporation by Reference

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference. Inaddition, the sequence listing submitted herewith is incorporated hereinby reference in its entirety. The specification and sequence listing ofeach of the following U.S. and PCT applications are herein incorporatedby reference in their entirety (filing dates shown in format“year-month-day” (yyyy-mm-dd)): Application No. 60/278,650 filed on 2001Mar. 27, application Ser. No. 09/950,082 filed on 2001 Sep. 12,application Ser. No. 09/950,083 filed on 2001 Sep. 12, application Ser.No. 60/306,171 filed on 19 Jul. 2001, application Ser. No. 09/833,245filed on 2001 Apr. 12, Application No. PCT/US01/11988 filed on 2001 Apr.12, Application No. 60/331,287 filed on 2001 Nov. 13, Application No.60/277,340 filed on 2001 Mar. 21, Application No. PCT/US00/06043 filedon 2000 Mar. 9, Application No. PCT/US00/06012 filed on 2000 Mar. 9,Application No. PCT/US00/06058 filed on 2000 Mar. 9, Application No.PCT/US00/06044 filed on 2000 Mar. 9, Application No. PCT/US00/06059filed on 2000 Mar. 9, Application No. PCT/US00/06042 filed on 2000 Mar.9, Application No. PCT/US00/06014 filed on 2000 Mar. 9, Application No.PCT/US00/06013 filed on 2000 Mar. 9, Application No. PCT/US00/06049filed on 2000 Mar. 9, Application No. PCT/US00/06057 filed on 2000 Mar.9, Application No. PCT/US00/06824 filed on 2000 Mar. 16, Application No.PCT/US00/06765 filed on 2000 Mar. 16, Application No. PCT/US00/06792filed on 2000 Mar. 16, Application No. PCT/US00/06830 filed on 2000 Mar.16, Application No. PCT/US00/06782 filed on 2000 Mar. 16, ApplicationNo. PCT/US00/06822 filed on 2000 Mar. 16, Application No. PCT/US00/06791filed on 2000 Mar. 16, Application No. PCT/US00/06828 filed on 2000 Mar.16, Application No. PCT/US00/06823 filed on 2000 Mar. 16, ApplicationNo. PCT/US00/06781 filed on 2000 Mar. 16, Application No. PCT/US00/07505filed on 2000 Mar. 22, Application No. PCT/US00/07440 filed on 2000 Mar.22, Application No. PCT/US00/07506 filed on 2000 Mar. 22, ApplicationNo. PCT/US00/07507 filed on 2000 Mar. 22, Application No. PCT/US00/07535filed on 2000 Mar. 22, Application No. PCT/US00/07525 filed on 2000 Mar.22, Application No. PCT/US00/07534 filed on 2000 Mar. 22, ApplicationNo. PCT/US00/07483 filed on 2000 Mar. 22, Application No. PCT/US00/07526filed on 2000 Mar. 22, Application No. PCT/US00/07527 filed on 2000 Mar.22, Application No. PCT/US00/07661 filed on 2000 Mar. 23, ApplicationNo. PCT/US00/07579 filed on 2000 Mar. 23, Application No. PCT/US00/07723filed on 2000 Mar. 23, Application No. PCT/US00/07724 filed on 2000 Mar.23, Application No. PCT/US00/14929 filed on 2000 Jun. 1, Application No.PCT/US00/07722 filed on 2000 Mar. 23, Application No. PCT/US00/07578filed on 2000 Mar. 23, Application No. PCT/US00/07726 filed on 2000 Mar.23, Application No. PCT/US00/07677 filed on 2000 Mar. 23, ApplicationNo. PCT/US00/07725 filed on 2000 Mar. 23, Application No. PCT/US00/09070filed on 2000 Apr. 6, Application No. PCT/US00/08982 filed on 2000 Apr.6, Application No. PCT/US00/08983 filed on 2000 Apr. 6, Application No.PCT/US00/09067 filed on 2000 Apr. 6, Application No. PCT/US00/09066filed on 2000 Apr. 6, Application No. PCT/US00/09068 filed on 2000 Apr.6, Application No. PCT/US00/08981 filed on 2000 Apr. 6, Application No.PCT/US00/08980 filed on 2000 Apr. 6, Application No. PCT/US00/09071filed on 2000 Apr. 6, Application No. PCT/US00/09069 filed on 2000 Apr.6, Application No. PCT/US00/15136 filed on 2000 Jun. 1, Application No.PCT/US00/14926 filed on 2000 Jun. 1, Application No. PCT/US00/14963filed on 2000 Jun. 1, Application No. PCT/US00/15135 filed on 2000 Jun.1, Application No. PCT/US00/14934 filed on 2000 Jun. 1, Application No.PCT/US00/14933 filed on 2000 Jun. 1, Application No. PCT/US00/15137filed on 2000 Jun. 1, Application No. PCT/US00/14928 filed on 2000 Jun.1, Application No. PCT/US00/14973 filed on 2000 Jun. 1, Application No.PCT/US00/14964 filed on 2000 Jun. 1, Application No. PCT/US00/26376filed on 2000 Sep. 26, Application No. PCT/US00/26371 filed on 2000 Sep.26, Application No. PCT/US00/26324 filed on 2000 Sep. 26, ApplicationNo. PCT/US00/26323 filed on 2000 Sep. 26, Application No. PCT/US00/26337filed on 2000 Sep. 26, Application No. PCT/US01/13318 filed on 2001 Apr.27, Application No. U.S. 60/124,146 filed on 1999 Mar. 12, ApplicationNo. U.S. 60/167,061 filed on 1999 Nov. 23, Application No. U.S.60/124,093 filed on 1999 Mar. 12, Application No. U.S. 60/166,989 filedon 1999 Nov. 23, Application No. U.S. 60/124,145 filed on 1999 Mar. 12,Application No. U.S. 60/168,654 filed on 1999 Dec. 3, Application No.U.S. 60/124,099 filed on 1999 Mar. 12, Application No. U.S. 60/168,661filed on 1999 Dec. 3, Application No. U.S. 60/124,096 filed on 1999 Mar.12, Application No. U.S. 60/168,622 filed on 1999 Dec. 3, ApplicationNo. U.S. 60/124,143 filed on 1999 Mar. 12, Application No. U.S.60/168,663 filed on 1999 Dec. 3, Application No. U.S. 60/124,095 filedon 1999 Mar. 12, Application No. U.S. 60/138,598 filed on 1999 Jun. 11,Application No. U.S. 60/168,665 filed on 1999 Dec. 3, Application No.U.S. 60/125,360 filed on 1999 Mar. 19, Application No. U.S. 60/138,626filed on 1999 Jun. 11, Application No. U.S. 60/168,662 filed on 1999Dec. 3, Application No. U.S. 60/124,144 filed on 1999 Mar. 12,Application No. U.S. 60/138,574 filed on 1999 Jun. 11, Application No.U.S. 60/168,667 filed on 1999 Dec. 3, Application No. U.S. 60/124,142filed on 1999 Mar. 12, Application No. U.S. 60/138,597 filed on 1999Jun. 11, Application No. U.S. 60/168,666 filed on 1999 Dec. 3,Application No. U.S. 60/125,359 filed on 1999 Mar. 19, Application No.U.S. 60/168,664 filed on 1999 Dec. 3, Application No. U.S. 60/126,051filed on 1999 Mar. 23, Application No. U.S. 60/169,906 filed on1999-12-10, Application No. U.S. 60/125,362 filed on 1999 Mar. 19,Application No. U.S. 60/169,980 filed on 1999 Dec. 10, Application No.U.S. 60/125,361 filed on 1999 Mar. 19, Application No. U.S. 60/169,910filed on 1999 Dec. 10, Application No. U.S. 60/125,812 filed on 1999Mar. 23, Application No. U.S. 60/169,936 filed on 1999 Dec. 10,Application No. U.S. 60/126,054 filed on 1999 Mar. 23, Application No.U.S. 60/169,916 filed on 1999 Dec. 10, Application No. U.S. 60/125,815filed on 1999 Mar. 23, Application No. U.S. 60/169,946 filed on 1999Dec. 10, Application No. U.S 60/125,358 filed on 1999 Mar. 19,Application No. U.S. 60/169,616 filed on 1999 Dec. 8, Application No.U.S. 60/125,364 filed on 1999 Mar. 19, Application No. U.S. 60/169,623filed on 1999 Dec. 8, Application No. U.S. 60/125,363 filed on 1999 Mar.19, Application No. U.S. 60/169,617 filed on 1999 Dec. 8, ApplicationNo. U.S. 60/126,502 filed on 1999 Mar. 26, Application No. U.S.60/172,410 filed on 1999 Dec. 17, Application No. U.S. 60/126,503 filedon 1999 Mar. 26, Application No. U.S 60/172,409 filed on 1999 Dec. 17,Application No. U.S. 60/126,505 filed on 1999 Mar. 26, Application No.U.S. 60/172,412 filed on 1999 Dec. 17, Application No. U.S. 60/126,594filed on 1999 Mar. 26, Application No. U.S. 60/172,408 filed on 1999Dec. 17, Application No. U.S. 60/126,511 filed on 1999 Mar. 26,Application No. U.S. 60/172,413 filed on 1999 Dec. 17, Application No.U.S. 60/126,595 filed on 1999 Mar. 26, Application No. U.S. 60/171,549filed on 1999 Dec. 22, Application No. U.S. 60/126,598 filed on 1999Mar. 26, Application No. U.S. 60/171,504 filed on 1999 Dec. 22,Application No. U.S. 60/126,596 filed on 1999 Mar. 26, Application No.U.S. 60/171,552 filed on 1999 Dec. 22, Application No. U.S. 60/126,600filed on 1999 Mar. 26, Application No. U.S. 60/171,550 filed on 1999Dec. 22, Application No. U.S. 60/126,501 filed on 1999 Mar. 26,Application No. U.S. 60/171,551 filed on 1999 Dec. 22, Application No.U.S. 60/126,504 filed on 1999 Mar. 26, Application No. U.S 60/174,847filed on 2000 Jan. 7, Application No. U.S. 60/126,509 filed on 1999 Mar.26, Application No. U.S. 60/174,853 filed on 2000 Jan. 7, ApplicationNo. U.S. 60/126,506 filed on 1999 Mar. 26, Application No. U.S.60/174,852 filed on 2000 Jan. 7, Application No. U.S. 60/242,710 filedon 2000 Oct. 25, Application No. U.S. 60/126,510 filed on 1999 Mar. 26,Application No. U.S. 60/174,850 filed on 2000 Jan. 7, Application No.U.S. 60/138,573 filed on 1999 Jun. 11, Application No. U.S 60/174,851filed on 2000 Jan. 7, Application No. U.S. 60/126,508 filed on 1999 Mar.26, Application No. U.S. 60/174,871 filed on 2000 Jan. 7, ApplicationNo. U.S. 60/126,507 filed on 1999 Mar. 26, Application No. U.S.60/174,872 filed on 2000 Jan. 7, Application No. U.S. 60/126,597 filedon 1999 Mar. 26, Application No. U.S. 60/174,877 filed on 2000 Jan. 7,Application No. U.S. 60/126,601 filed on 1999 Mar. 26, Application No.U.S. 60/154,373 filed on 1999 Sep. 17, Application No. U.S. 60/176,064filed on 2000 Jan. 14, Application No. U.S. 60/126,602 filed on 1999Mar. 26, Application No. U.S. 60/176,063 filed on 2000 Jan. 14,Application No. U.S. 60/128,695 filed on 1999 Apr. 9, Application No.U.S. 60/176,052 filed on 2000 Jan. 14, Application No. U.S. 60/128,696filed on 1999 Apr. 9, Application No. U.S. 60/176,069 filed on 2000 Jan.14, Application No. U.S. 60/128,703 filed on 1999 Apr. 9, ApplicationNo. U.S. 60/176,068 filed on 2000 Jan. 14, Application No. U.S.60/128,697 filed on 1999 Apr. 9, Application No. U.S. 60/176,929 filedon 2000 Jan. 20, Application No. U.S. 60/128,698 filed on 1999 Apr. 9,Application No. U.S. 60/176,926 filed on 2000 Jan. 20, Application No.U.S. 60/128,699 filed on 1999 Apr. 9, Application No. U.S. 60/177,050filed on 2000 Jan. 20, Application No. U.S. 60/128,701 filed on 1999Apr. 9, Application No. U.S. 60/177,166 filed on 2000 Jan. 20,Application No. U.S. 60/128,700 filed on 1999 Apr. 9, Application No.U.S. 60/176,930 filed on 2000 Jan. 20, Application No. U.S. 60/128,694filed on 1999 Apr. 9, Application No. U.S. 60/176,931 filed on 2000 Jan.20, Application No. U.S. 60/128,702 filed on 1999 Apr. 9, ApplicationNo. U.S. 60/177,049 filed on 2000 Jan. 20, Application No. U.S.60/138,629 filed on 1999 Jun. 11, Application No. U.S. 60/138,628 filedon 1999 Jun. 11, Application No. U.S. 60/138,631 filed on 1999 Jun. 11,Application No. U.S. 60/138,632 filed on 1999 Jun. 11, Application No.U.S. 60/138,599 filed on 1999 Jun. 11, Application No. U.S. 60/138,572filed on 1999 Jun. 11, Application No. U.S. 60/138,625 filed on 1999Jun. 11, Application No. U.S. 60/138,633 filed on 1999 Jun. 11,Application No. U.S. 60/138,630 filed on 1999 Jun. 11, Application No.U.S. 60/138,627 filed on 1999 Jun. 11, Application No. U.S. 60/155,808filed on 1999 Sep. 27, Application No. U.S. 60/155,804 filed on 1999Sep. 27, Application No. U.S. 60/155,807 filed on 1999 Sep. 27,Application No. U.S. 60/155,805 filed on 1999 Sep. 27, Application No.U.S. 60/155,806 filed on 1999 Sep. 27, Application No. U.S. 60/201,194filed on 2000 May 2, Application No. U.S. 60/212,142 filed on 2000 Jun.16

1. Use of a polypeptide for the preparation of a diagnostic orpharmaceutical composition for diagnosing or treating diabetes orconditions related to diabetes, wherein said polypeptide comprises anamino acid sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a full length polypeptide of SEQ ID NO:Y ora full length polypeptide encoded by the cDNA Clone ID in ATCC DepositNo:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (b) apredicted secreted form of SEQ ID NO:Y or a secreted form of thepolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (c) apolypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded bythe cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y asreferenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (e) a polypeptide domain of SEQ IDNO:Y as referenced in Table 1B; (f) a polypeptide domain of SEQ ID NO:Yas referenced in Table 2; and (g) a predicted epitope of SEQ ID NO:Y asreferenced in Table 1B.
 2. Use of the polypeptide of claim 1, whereinsaid wherein said polypeptide comprises a heterologous amino acidsequence.
 3. Use of a polypeptide for the preparation of a diagnostic orpharmaceutical composition for diagnosing or treating diabetes orconditions related to diabetes, wherein said polypeptide comprises anamino acid sequence selected from the group consisting of: (a) a fulllength polypeptide of SEQ ID NO:Y or a full length polypeptide encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A; (b) a predicted secreted form of SEQ ID NO:Yor a secreted form of the polypeptide encoded by the cDNA Clone ID inATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table1A; (c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragmentencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (d) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A,wherein said fragment has biological activity; (e) a polypeptide domainof SEQ ID NO:Y as referenced in Table 1B; (f) a polypeptide domain ofSEQ ID NO:Y as referenced in Table 2; and (g) a predicted epitope of SEQID NO:Y as referenced in Table 1B.
 4. Use of the polypeptide of claim 3,wherein said polypeptide comprises a heterologous amino acid sequence.5. Use of an antibody or fragment thereof for the preparation of adiagnostic or pharmaceutical composition for diagnosing or treatingdiabetes or conditions related to diabetes, wherein said antibody orfragment thereof binds a polypeptide comprising an amino acid sequenceat least 95% identical to a sequence selected from the group consistingof: (a) a full length polypeptide of SEQ ID NO:Y or a full lengthpolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A, wherein said fragment has biologicalactivity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table1B; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2;and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B. 6.Use of an antibody or fragment thereof for the preparation of adiagnostic or pharmaceutical composition for diagnosing or treatingdiabetes or conditions related to diabetes, wherein said antibody orfragment thereof binds a polypeptide selected from the group consistingof: (a) a full length polypeptide of SEQ ID NO:Y or a full lengthpolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A, wherein said fragment has biologicalactivity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table1B; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2;and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B. 7.Use of a nucleic acid molecule for the preparation of a diagnostic orpharmaceutical composition for diagnosing or treating diabetes orconditions related to diabetes, wherein said nucleic acid moleculecomprises a polynucleotide sequence at least 95% identical to a sequenceselected from the group consisting of: (a) a polynucleotide fragment ofSEQ ID NO:X as referenced in Table 1A; (b) a polynucleotide encoding afull length polypeptide of SEQ ID NO:Y or a full length polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polynucleotide encoding apredicted secreted form of SEQ ID NO:Y or a secreted form of thepolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (d) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (e) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (f) a polynucleotide encoding apolypeptide domain of SEQ ID NO:Y as referenced in Table 1B; (g) apolynucleotide encoding a polypeptide domain of SEQ ID NO:Y asreferenced in Table 2; and (h) a polynucleotide encoding a predictedepitope of SEQ ID NO:Y as referenced in Table 1B.
 8. Use of the nucleicacid molecule of claim 7, wherein said nucleic acid molecule comprises aheterologous polynucleotide sequence.
 9. Use of a nucleic acid moleculefor the preparation of a diagnostic or pharmaceutical composition fordiagnosing or treating diabetes or conditions related to diabetes,wherein said nucleic acid molecule comprises a polynucleotide sequenceselected from the group consisting of: (a) a polynucleotide fragment ofSEQ ID NO:X as referenced in Table 1A; (b) a polynucleotide encoding afull length polypeptide of SEQ ID NO:Y or a full length polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polynucleotide encoding apredicted secreted form of SEQ ID NO:Y or a secreted form of thepolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (d) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (e) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (f) a polynucleotide encoding apolypeptide domain of SEQ ID NO:Y as referenced in Table 1B; (g) apolynucleotide encoding a polypeptide domain of SEQ ID NO:Y asreferenced in Table 2; and (h) a polynucleotide encoding a predictedepitope of SEQ ID NO:Y as referenced in Table 1B.
 10. Use of the nucleicacid molecule of claim 9, wherein said nucleic acid molecule comprises aheterologous polynucleotide sequence.
 11. Use of an agonist orantagonist for the preparation of a pharmaceutical composition fortreating diabetes or conditions related to diabetes, wherein saidagonist or antagonist binds a polypeptide comprising an amino acidsequence at least 95% identical to a sequence selected from the groupconsisting of: (a) a full length polypeptide of SEQ ID NO:Y or a fulllength polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A, wherein said fragment has biologicalactivity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table1B; (f). a polypeptide domain of SEQ ID NO:Y as referenced in Table 2;and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.12. Use of an agonist or antagonist for the preparation of apharmaceutical composition for treating diabetes or conditions relatedto diabetes, wherein said agonist or antagonist binds a polypeptideselected from the group consisting of: (a) a full length polypeptide ofSEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID inATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table1A; (b) a predicted secreted form of SEQ ID NO:Y or a secreted form ofthe polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (c) apolypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded bythe cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y asreferenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (e) a polypeptide domain of SEQ IDNO:Y as referenced in Table 1B; (f) a polypeptide domain of SEQ ID NO:Yas referenced in Table 2; and (g) a predicted epitope of SEQ ID NO:Y asreferenced in Table 1B.
 13. A polypeptide comprising an amino acidsequence at least 95% identical to a sequence selected from the groupconsisting of: (a) a full length polypeptide of SEQ ID NO:Y or a fulllength polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A, wherein said fragment has biologicalactivity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table1B; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2;and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.14. The polypeptide of claim 13, wherein said polypeptide comprises aheterologous amino acid sequence.
 15. Use of the polypeptide of claim 13for identifying a binding partner comprising: (a) contacting thepolypeptide of claim 13 with a binding partner; and (b) determiningwhether the binding partner increases or decreases activity of thepolypeptide.
 16. A polypeptide comprising an amino acid sequenceselected from the group consisting of: (a) a full length polypeptide ofSEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID inATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table1A; (b) a predicted secreted form of SEQ ID NO:Y or a secreted form ofthe polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (c) apolypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded bythe cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y asreferenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (e) a polypeptide domain of SEQ IDNO:Y as referenced in Table 1B; (f) a polypeptide domain of SEQ ID NO:Yas referenced in Table 2; and (g) a predicted epitope of SEQ ID NO:Y asreferenced in Table 1B.
 17. The polypeptide of claim 16, wherein saidpolypeptide comprises a heterologous polypeptide sequence.
 18. Use ofthe polypeptide of claim 16 for identifying a binding partnercomprising: (a) contacting the polypeptide of claim 16 with a bindingpartner; and (b) determining whether the binding partner increases ordecreases activity of the polypeptide.
 19. An antibody or fragmentthereof that binds a polypeptide comprising an amino acid sequence atleast 95% identical to a sequence selected from the group consisting of:(a) a full length polypeptide of SEQ ID NO:Y or a full lengthpolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ IDNO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A, wherein said fragment has biologicalactivity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table1B; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2;and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.20. An antibody or fragment thereof that binds a polypeptide selectedfrom the group consisting of: (a) a full length polypeptide of SEQ IDNO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (b)a predicted secreted form of SEQ ID NO:Y or a secreted form of thepolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (c) apolypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded bythe cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y asreferenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (e) a polypeptide domain of SEQ IDNO:Y as referenced in Table 1B; (f) a polypeptide domain of SEQ ID NO:Yas referenced in Table 2; and (g) a predicted epitope of SEQ ID NO:Y asreferenced in Table 1B.
 21. A nucleic acid molecule comprising apolynucleotide sequence at least 95% identical to a sequence selectedfrom the group consisting of: (a) a polynucleotide fragment of SEQ IDNO:X as referenced in Table 1A; (b) a polynucleotide encoding a fulllength polypeptide of SEQ ID NO:Y or a full length polypeptide encodedby the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Yas referenced in Table 1A; (c) a polynucleotide encoding a predictedsecreted form of SEQ ID NO:Y or a secreted form of the polypeptideencoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQID NO:Y as referenced in Table 1A; (d) a polynucleotide encoding apolypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded bythe cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y asreferenced in Table 1A; (e) a polynucleotide encoding a polypeptidefragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNAClone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referencedin Table 1A, wherein said fragment has biological activity; (f) apolynucleotide encoding a polypeptide domain of SEQ ID NO:Y asreferenced in Table 1B; (g) a polynucleotide encoding a polypeptidedomain of SEQ ID NO:Y as referenced in Table 2; and (h) a polynucleotideencoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.22. The nucleic acid molecule of claim 21, wherein said nucleic acidmolecule comprises a heterologous polynucleotide sequence.
 23. Arecombinant vector comprising the nucleic acid molecule of claim
 21. 24.A recombinant vector comprising the nucleic acid molecule of claim 22.25. A recombinant host cell comprising the recombinant vector of claim23.
 26. A recombinant host cell comprising the recombinant vector ofclaim
 24. 27. A nucleic acid molecule comprising a polynucleotidesequence selected from the group consisting of: (a) a polynucleotidefragment of SEQ ID NO:X as referenced in Table 1A; (b) a polynucleotideencoding a full length polypeptide of SEQ ID NO:Y or a full lengthpolypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (c) apolynucleotide encoding a predicted secreted form of SEQ ID NO:Y or asecreted form of the polypeptide encoded by the cDNA Clone ID in ATCCDeposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d)a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A; (e) apolynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or apolypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Zcorresponding to SEQ ID NO:Y as referenced in Table 1A, wherein saidfragment has biological activity; (f) a polynucleotide encoding apolypeptide domain of SEQ ID NO:Y as referenced in Table 1B; (g) apolynucleotide encoding a polypeptide domain of SEQ ID NO:Y asreferenced in Table 2; and (h) a polynucleotide encoding a predictedepitope of SEQ ID NO:Y as referenced in Table 1B.
 28. The nucleic acidmolecule of claim 27, wherein said nucleic acid molecule comprises aheterologous polynucleotide sequence.
 29. A recombinant vectorcomprising the nucleic acid molecule of claim
 27. 30. A recombinantvector comprising the nucleic acid molecule of claim
 28. 31. Arecombinant host cell comprising the recombinant vector of claim
 29. 32.A recombinant host cell comprising the recombinant vector of claim 30.